Workshop
30-31st May 2012

Research Instruments
Welcome & Introductions
Rob Watkins
UK/IRE Business & Sales Manager

Martyn Selley
UK/IRE Sales Manager

Martin Mankee
Int...
Welcome to Cornwall
Wednesday
•

Day 1 Morning

DAY 1: 8.00 – 9.30 Reception
The office will be open and able to receive trainees for Tea /Cof...
Day 1 Afternoon
•

13.45 – 15.45
–

•
•

Micromanipulation Continued

Practical session on the use of micromanipulators - ...
Day 2 Morning
•

Thursday

•
•
•

Day 2
8.30 – 9.00
9.00 – 10.00

Hotel Transfers to RI Training Suite
Meeting Start - Man...
Day 2 Afternoon
•

13.15 – 13.45

Manipulation and Biopsy II

Further hands on practical time and practice on the latest a...
How is this session going to work?
•
•
•
•

Please Remember this is designed to be a practical workshop
throughout
There w...
Learning Objectives Today
Today you will:
 Understand the fundamentals of the modern microscope used in the
IVF process
...
ACE SECTION - MICROSCOPY
• With the aim of a diagram describe the optical principles of a stereo
microscope
• With the aid...
Microscopy for IVF
Brief Background
The optical microscope, often referred to as the "light microscope", is a type of
microscope which uses v...
Introduction to Our Limitations
1. Physics – Its limitations on us in IVF
Any consideration of microscopy must begin with ...
2. Biology – Its limitations on us in IVF
•

There are only 2 things that the eye can see. ???
The CONES respond to bright...
Wavelength is the property we see as colour

The human eye sees (RGB) light in equal amounts as white light. All the other...
Please bear in mind not all light is the same…
Coherence
Coherent waves are all of the same frequency and all the
waves ar...
Wavelength

Colour always arises from
light, there is no other source
All Colour super impose depending on the
predominant...
3. Intensity - Represented by Amplitude

So we can only see our specimen if it has a different
1.COLOUR (wavelength) or
2....
How do we see objects that are not
light sources themselves ?
• There are different processes, which happen when light mee...
Colour Shift
• A colour phenomenon – An everyday experience
– Clothes bought in the shop often look different when
brought...
Summary of Light in Our Workshops
here at Research Instruments
• The light that reaches our eye consists of many different...
COFFEE!
Summary of Session One
• In this work shop we are using white light as you do in the lab
• White light is made up of many ...
Light Microscope
Schematic diagram
of a light
microscope
outfitted for
photomicrography
And what’s the difference between
...
Jargon
•

Resolution- Resolution can be defined as the least distance between 2 points at
which they can still be recogniz...
What is Resolution ?
The ability to visually separate dots

Every specimen detail
that is illuminated creates
a so called ...
Resolution

Note: If the incident radiation contains several wavelengths, each
wavelength deviates through a specific angl...
• The smaller the Airy disks,
the higher the resolution in
an image. Objectives which
have a higher numerical
aperture pro...
Resolution and Limit of Resolution
Limit of Resolution for a light microscope is 1000x Magnification
Wavelength versus Resolution
Wavelength
(Nanometers)

Resolution
(Micrometers)

360

0.19

400

0.21

450

0.24

500

0.26...
Electron Microscopes – remember our limits…
•
•

•
•
•
•
•

Benefits are that they do not use light but fire electrons tha...
Jargon
• Numerical ApertureThis indicates the resolving ability of an objective. Larger N.A.= Greater
resolution and also ...
Numerical Aperture N.A.
NA = n Sin
Numerical
Aperture

Immersion
Media
Refractive
Index

Angle of the
Cone of
illumination...
Jargon
•

Polarisation – The rotation and direction of the light wave expressed as its
orientation

•

Working Distance- (...
Orientation and Polarisation
(end-on view)

(end-on view)

(end-on view)

(end-on view)
Polarised light in Microscopy and
Illumination Techniques

Huygens’ Principle and
Diffraction Pattern
Numerical Aperture (NA) and Working Distance (WD)
sample

WD

WD

WD

Resolution
Let’s not get too involved here…
Far away… NA 0.2

ABDFGRG
BNHYUKF
LGMNJDE
GFKGLWL
KLIIURRM

Closer… NA 0.9
ABDFGRG
BNHYUK...
Jargon
•

Chromatic Aberration- When white light passes through a convex lens, the
colours split and focus at different po...
Aberrations
Chromatic Aberration

Coma
B
A

Spherical Aberration

Astigmatism
C

On-Axis

S

T

Off-Axis
Chromatic aberration
Objectives
Objective Lens Nomenclature

Red = 4x
Yellow = 10x
Green = 20x
Blue = 40 x
Objective Lens Types
Types of Objectives / Heads
•

No designation – Dry Objective / Non Oil

•

Infinity – High quality infinity corrected

•
...
High Mag. Objectives & Correction Collar
•
•
•

Corrects for Spherical Aberration
Caused by variations in material thickne...
Light-Gathering Power
When we alter the light beam we affect the brightness. The more we do,
the less light we have to pus...
Eyepieces
•
•
•
•
•

•
•

Typically 10 x sometimes 15x Magnification
These form an integral part of the magnification of t...
COFFEE!
LUNCH & DINNER
(Weather Dependant)
• Dinner Tonight :
– Choices
• Sea Food
• Combination
• Beach
• Italian
LUNCH

Menu cho...
Visualisation Techniques
•

The job of the light based equipment (Light, polariser, condenser, HMC etc) is to
present the ...
Visualisation Techniques
 Brightfield – Good for stained specimens
 Darkfield – True colour against black background
 P...
Visualisation Techniques
Brightfield

Phase Contrast

DIC
Nomarski
Visualisation Techniques
Brightfield

Darkfield
What is Darkfield then....
• Stars in the sky....
– We cant see the stars in daylight on a clear day yet we
know they are ...
Bright field
IMAGE

Objective

Sample
Darkfield Cont.
• Therefore we can see a particle or sample in front of a black
background even when the particle itself i...
Diagram of a dedicated
Bright field – Dark field system
Oblique Illumination
• The simplest way to achieve a contrast
method that results in a relief-like image (2D/3D)
is by usi...
Limits of Oblique illumination
The contrast itself is produced by the complete thickness of
the specimen and the resolutio...
Polariser in Microscopy
• Below the condenser, a
circular polarizer is placed
on the light exit port of the
microscope. Th...
HMC

• Hoffman Modulation Contrast - Hoffman modulation
contrast is an oblique illumination technique that
enhances contra...
Hoffman
•

The Hoffman Modulation Contrast system
is designed to increase visibility and
contrast in unstained and living ...
Principles of light path in Hoffman
Visualisation Techniques ii
Hoffman Modulation Contrast (HMC)
Condenser
• Above the stage, a condenser
with rotating turret is utilized to
hold the remaining components
of the Hoffman ...
HOFFMAN MODULATION CONTRAST i
- HMC
Objective

Condenser

• The modulator has three zones that are depicted in Figure 2: a small, dark
zone near the periphery...
DIC / Nomarski
• An excellent mechanism for rendering contrast in transparent specimens,
differential interference contras...
Phase Contrast
•

Light that is travelling through part of a specimen and is not absorbed by amplitude
objects will not pr...
Diagram

Principles of DIC / Nomarski
Visualisation Techniques
Phase Contrast
Alignment of Visualisation Techniques
- off axis aberration in phase contrast
Objective for Phase Microscopy
Diagram

Principles of DIC / Nomarski
Visualisation Techniques
Differential Interference Contrast (DIC)
Alignment of Visualisation Techniques
- off axis aberration in phase contrast
HARDWARE MICROSCOPY
Microscopes Stereo and Compound
•

Compound Microscopes use two lenses to focus light. 1st lens is the...
Light Microscope
Schematic diagram
of a light
microscope
outfitted for
photomicrography
And what’s the difference between
...
Stereo Microscopes
•

Variety of Illumination options

•

Transmitted Brightfield, Darkfield, Oblique and Polarised Light
...
Stereo zooms in IVF
Typically either mounted in a class II Cabinet with the cabinet companies light
source or as a stand a...
Stereo Microscopes
•

2 separate optical paths at an angle to each other

•

Brain merges the 2 images to give a 3D image ...
Greenhough Optics

• 10 Degree
Converging light
path
• Great depth of
focus

To see objects within a
microscope in a simil...
Galilean Optics

•Parallel Optics
•Allows build up of
other accessories
Inverted Microscopes
•

Predominately used for looking at specimens in suspension- Live cells – ICSI

•

Requires Long wor...
Inverted Scopes in IVF
•
•
•

•

Typically only found with micro
manipulators in IVF lab
Featured here with the Integra
ba...
Upright Microscopes
•

Samples on glass slides (Andrology)

•

Small Working distance

•

Variety of Contrast techniques u...
The MicroscopeWhat is Important?
•

Location – Ideally in an area with few disturbances

•

Avoid direct lighting - not ne...
Imaging in the IVF Laboratory
•

The image from an optical microscope can be captured by normal light-sensitive
cameras to...
Beam Splitter / Prism
CCD Camera
•
•

•

Charged Couple Device - CCD
Cameras are have microscope specific mountings
on them which often feature ...
Cleaning and Care
• Never touch the lenses with your fingers. Your body
produces an oil that smudges the glass
• Oil can e...
Break
Manipulators

START OF OUR PRACTICAL ICSI SESSIONS
Please find yourself a system.
Controls
Course Control
x and y control
10 micron resolution
4mm x and y travel
Stage

Fine Control
XYZ movement

Sub-micr...
PL30 and MPH &Tool Holders
Toolholder
angle adjustment- 15 to 40
degrees. Vertical, axial and
rotational movements
MPH
Mic...
Tool holders
• The tool holders provide the simplest and quickest method ever for
setting-up micropipettes. The tool holde...
Pipette Challenge
Time to practice with the rigs
Spend time with the optical
path arrangements of each
scope

Understand w...
What we are looking at is not always what it is and
sometimes it is very well hidden!

The art of good IVF
microscopy is e...
Wednesday
•

Day 1 Morning

DAY 1: 8.00 – 9.30 Reception
The office will be open and able to receive trainees for Tea /Cof...
Day 1 Afternoon
•

13.45 – 15.45
–

•
•

Micromanipulation Continued

Practical session on the use of micromanipulators - ...
END OF DAY 1
•
•
•
•

Transfer to Hotels with Rob & Martyn
Confirmation of Hotel
Pick up to be agreed with each of you.
Pl...
Tomorrow Morning
• 8.30- 8.45 am PICK UP at Hotels for those who need it
– Train transfers in the afternoon will be made d...
Day 2
Day 2 Morning
•

Thursday

•
•
•

Day 2
8.30 – 9.00
9.00 – 10.00

I NEED A FAVOUR...
Hotel Transfers to RI Training Suite
...
Day 2 Afternoon
•

13.15 – 13.45

Manipulation and Biopsy II

Further hands on practical time and practice on the latest a...
Integra Ti components
Syringes
Temperature variation with objectives
40.0

ITO Heated Stage with Objectives (4x 10x 20x)
Bourn Hall 13/04/12
Ref 1
10x ro...
Parfocal Alignment
Alignment of Olympus Relief or
Hoffman Modulation
Biopsy Session -
IMSI Session
Microscope Workshop
In Association with Research Instruments
Nikon, & Olympus Microscopes
Stay in touch
rwatkins@research-instruments.com
01326370606 / 07795438952

Like us on Facebook
Follow us on Twitter @RI_Li...
Thank you!
Microscopy and Micromanipulation for UK/IRE Embryology Workshop - Research Instruments - RIUK - Rob Watkins 2012
Microscopy and Micromanipulation for UK/IRE Embryology Workshop - Research Instruments - RIUK - Rob Watkins 2012
Microscopy and Micromanipulation for UK/IRE Embryology Workshop - Research Instruments - RIUK - Rob Watkins 2012
Microscopy and Micromanipulation for UK/IRE Embryology Workshop - Research Instruments - RIUK - Rob Watkins 2012
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Microscopy and Micromanipulation for UK/IRE Embryology Workshop - Research Instruments - RIUK - Rob Watkins 2012

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Presentation given to Students at the RIUK Embryology Workshop in 2012. Based at our training facility at Reseach Instruments HQ in Falmouth, Cornwall, UK. This selection of slides is taken from the 2 day sessions that I ran throughout that year. It covers most of the areas that we feel are relevent to the users of both the microscopes and micromanipulators (Integra Ti) that they have in their clinics.

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Microscopy and Micromanipulation for UK/IRE Embryology Workshop - Research Instruments - RIUK - Rob Watkins 2012

  1. 1. Workshop 30-31st May 2012 Research Instruments
  2. 2. Welcome & Introductions Rob Watkins UK/IRE Business & Sales Manager Martyn Selley UK/IRE Sales Manager Martin Mankee International Service Manager Directors: Bill, Justin & David
  3. 3. Welcome to Cornwall
  4. 4. Wednesday • Day 1 Morning DAY 1: 8.00 – 9.30 Reception The office will be open and able to receive trainees for Tea /Coffee reception (for early arrivals!) til 9.30am Please arrive each day in advance of the scheduled start time so we can start promptly Some attendees will have arranged for RI to pick them up from local accommodation - This will be carried out at this time Location: RI Head Office • 9.30 – 10.15 – Directors and UK Managers welcome Introductions and reception with a tour of the facilities and transfer down to the training Suite at Site B Location: Boardroom RI Head Office • 10.15 – 10.30 – Introduction and Training aims for the practical session Training aims and individual itinerary discussion Familiarisation with the format and availability of the range of equipment Location: Training Suite Site B. The rest of the sessions will take place here • 10.30 - 11.30 Session 1 Microscopy – Basics (Nikon, Olympus, Leica) Fundamentals of microscopy work and sample illumination • • 11.30 – 11.45 Short Break 11.45 – 12.15 Session 2 Microscopy – IVF Principles for Microscopy in the IVF application including proper set up of Condenser Illumination, Microscope and Optics • 12.15 – 12.45 Fundamentals of Micromanipulation Integra and Manipulation techniques • 12.45 – 13.45 Lunch – Off Site Location
  5. 5. Day 1 Afternoon • 13.45 – 15.45 – • • Micromanipulation Continued Practical session on the use of micromanipulators - including but not exclusively: • Pipette selection and set-up • Quick set up guide of Pipettes on stage • Understanding the TDU • Understanding the PL30 and MPH • Overview of the HUD and Heated stages – Limitations and Benefits of ITO surfaces • Trouble shooting for common errors in set up in an IVF Lab 15.45 – 16.00 16.00 – 16.45 Short Break System and Pipette set up challenge A fun recap on the practical learning's of the day’s study • 16.45 – 17.00 Close of Day Key learning's and Transfers to accommodation • 19.30 - Late Evening Events – Smart Casual Dinner and Drinks in a seasonally appropriate venue Details to be confirmed in the morning with the Students
  6. 6. Day 2 Morning • Thursday • • • Day 2 8.30 – 9.00 9.00 – 10.00 Hotel Transfers to RI Training Suite Meeting Start - Manipulator Mania A fun, practical challenge for two teams to enhance and develop both the understanding and use of the main assemblies and tools in micromanipulation learnt on day 1 and applied in these day’s sessions • 10.00 – 11.00 Introduction to Advanced Techniques Biopsy and IMSI use in IVF with the RI Saturn ACTIVE and RI IMSI • • 11.00 – 11.15 11.15 – 12.45 Short Break Manipulation and Biopsy I Instruction on using laser assisted techniques and software Open session and material available for a wide range of techniques either building on the previous days learning's or the start of new skills on more advances equipment • 12.45 – 13.15 Lunch - On site catering
  7. 7. Day 2 Afternoon • 13.15 – 13.45 Manipulation and Biopsy II Further hands on practical time and practice on the latest available ICSI/PGD and IMSI equipment Throughout the sessions and during time when other equipment is taken up with other students instruction for use of the following environmental monitors will be available • 13.45 – 1400 • • Short Break and Sugar Cornwall’s best tradition to keep us going 14.00 – 15.15 Individual Development Session – The Final Session! Individual itinerary focus for students to cover any aspect of the two days as they and the trainer see fit There will be almost 1-1 coverage of each aspect of the two days learning at each work areas with a wealth of RI trainers available for support • 15.15 – 15.30 Close of Workshop and Thanks Transfer to accommodation or trains and departures • 15.30 – Late After Hours The RI UK team will be available in the training suite for a further hour for those staying on Anyone staying another night in Falmouth are welcome to join us again for an evening out
  8. 8. How is this session going to work? • • • • Please Remember this is designed to be a practical workshop throughout There will be a little “Death by PowerPoint” Although I need to show you something on the board, I believe in “see one, do one” teaching methods There is a reason that Microscopes do not appear too often in your Embryology text books! Matchsticks are available!
  9. 9. Learning Objectives Today Today you will:  Understand the fundamentals of the modern microscope used in the IVF process  Undertake training of stereo and inverted microscopes and the contrast method for equipment you either currently have or are likely to be exposed to.  Take away useful day to day operational and set up procedures of the Integra TI and associated hardware An Industry Certificate will be provided. This will be sent to you from RI head office after the session.
  10. 10. ACE SECTION - MICROSCOPY • With the aim of a diagram describe the optical principles of a stereo microscope • With the aid of a diagram describe the components of a compound microscope • When are the following used – i. Brightfield – ii Darkfield • When might oil immersion be used and why • Using a diagram describe the principles of phase contrast, Hoffman and Nomarski (DIC) • Use scopes in your lab • List procedure for cleaning • List the equipment in your lab and reason for use.
  11. 11. Microscopy for IVF
  12. 12. Brief Background The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were designed around 1600. Basic optical microscopes can be very simple, although there are many complex designs which aim to improve resolution and sample contrast. Historically optical microscopes were easy to develop and are popular because they use visible light so the sample can be directly observed by the eye.
  13. 13. Introduction to Our Limitations 1. Physics – Its limitations on us in IVF Any consideration of microscopy must begin with an understanding of how we perceive light and how light interacts with matter. Light has both a particle and a wave nature. For most of this discussion, it is the wave (sine wave) property of light which is of interest to us. It also travels in straight lines.
  14. 14. 2. Biology – Its limitations on us in IVF • There are only 2 things that the eye can see. ??? The CONES respond to bright light and mediate high-resolution COLOUR vision during daylight illumination (also called photopic vision). Three types sensitive to (RGB) primary colour The RODS are saturated at daylight levels. Rods respond to INTENSITY and mediate lower-resolution, monochromatic vision under very low levels of illumination (called scotopic vision).
  15. 15. Wavelength is the property we see as colour The human eye sees (RGB) light in equal amounts as white light. All the other colours are generated by stimulation of the three different types of cones. Black is the absence of colour and / or intensity.
  16. 16. Please bear in mind not all light is the same… Coherence Coherent waves are all of the same frequency and all the waves are in phase (that is, all the waves are "up" at the same point). Laser light is coherent. A green light can be monochromatic but still be incoherent because the waves are in random phase. Actually, an incoherent wave would have some dispersion, although it might be quite narrow, but it can't be too narrow, or it would be coherent for all intents and purposes. White light is incoherent both because the phase of the waves are random and because white light is made of many different frequencies simultaneously.
  17. 17. Wavelength Colour always arises from light, there is no other source All Colour super impose depending on the predominant wavelength palette
  18. 18. 3. Intensity - Represented by Amplitude So we can only see our specimen if it has a different 1.COLOUR (wavelength) or 2.INTENSITY (amplitude) …than its background …In IVF, our samples are very small and have no colour.
  19. 19. How do we see objects that are not light sources themselves ? • There are different processes, which happen when light meets physical matter. They are: Reflection:- is a change in direction at an interface Diffraction:- is the bending of a wave around objects or the spreading after passing through a gap (slit) Refraction :- is the change in direction of a wave due to a change in its speed Absorption:the way by which the energy is taken up in matter
  20. 20. Colour Shift • A colour phenomenon – An everyday experience – Clothes bought in the shop often look different when brought out into day light This can be observed in the microscope if we focus on an object and increase the power slowly The image observed will change from a yellow-red to a more bluish and then very bright image
  21. 21. Summary of Light in Our Workshops here at Research Instruments • The light that reaches our eye consists of many different colours, and different light sources produce a mix of these • Our vision gives us only a partial view of reality • For effective imaging the microscopist must be aware of the complexity of the world of light • There is a variety of light sources, objectives and filters to enable and assemble the microscope for a purpose • Several hundred years of development of optics have managed to hide come of the complexity from the regular use
  22. 22. COFFEE!
  23. 23. Summary of Session One • In this work shop we are using white light as you do in the lab • White light is made up of many different types of wavelength (colours) • Light is seen by its colour and / or its intensity – in relation to its background • We are limited by what our eyes can see, although the very perception of an image is a remarkable process using complex structures that both catch ,process then interpret them
  24. 24. Light Microscope Schematic diagram of a light microscope outfitted for photomicrography And what’s the difference between this and the invert ?
  25. 25. Jargon • Resolution- Resolution can be defined as the least distance between 2 points at which they can still be recognized as 2 separate entities • For the eye, this is 70 microns, when the object is 250mm away • For light microscopy, this is 0.24 microns • Contrast-The phenomena that allows you to distinguish relevant information from irrelevant. Either by colour or intensity • Contrast and Resolution are inversely proportional
  26. 26. What is Resolution ? The ability to visually separate dots Every specimen detail that is illuminated creates a so called diffraction pattern or airy disk. When two details within the specimen are closely together we can only see them separated If the two central spots are not too close together and the airy disks themselves do not overlap
  27. 27. Resolution Note: If the incident radiation contains several wavelengths, each wavelength deviates through a specific angle, the shorter the wavelength the less deviation and thus the better the resolution
  28. 28. • The smaller the Airy disks, the higher the resolution in an image. Objectives which have a higher numerical aperture produce smaller airy disks from the same specimen detail than low NA objectives
  29. 29. Resolution and Limit of Resolution Limit of Resolution for a light microscope is 1000x Magnification
  30. 30. Wavelength versus Resolution Wavelength (Nanometers) Resolution (Micrometers) 360 0.19 400 0.21 450 0.24 500 0.26 550 0.29 600 0.32 650 0.34 700 0.37
  31. 31. Electron Microscopes – remember our limits… • • • • • • • Benefits are that they do not use light but fire electrons that allow us to see detail smaller than wavelengths of light itself Therefore their Limit of Resolution (LOR)is much higher (A light microscopes LOR=1000x) Limit of resolution is near 1 million times therefore magnification of 350,000x is possible with Electron Microscopes - Significantly better than what we use TEMs – Transmission (2D) through the samples SEMs – Scanning (3D) bounce off the samples Cells have to be stationary and preparation and scanning actually kills denatures them We have to make a compromise with IVF samples
  32. 32. Jargon • Numerical ApertureThis indicates the resolving ability of an objective. Larger N.A.= Greater resolution and also brighter fluorescence signal. However larger N.A.= less depth of field and shorter working distance. NA= nSinA, where n= refractive index of medium and A is the half angle at which light enters the objective. 1 Shorter Working distance Depth of Field NA 0 Resolution
  33. 33. Numerical Aperture N.A. NA = n Sin Numerical Aperture Immersion Media Refractive Index Angle of the Cone of illumination The refractive index of the imaging medium is criticalof immersion mediaworking N.A. can never be higher than the refractive index in determining the numerical aperture of a microscope objective. A dramatic increase in numerical aperture is observed when the objective is designed to operate with an immersion medium such as oil, glycerin, or water between the front lens and the specimen cover glass
  34. 34. Jargon • Polarisation – The rotation and direction of the light wave expressed as its orientation • Working Distance- (W.D)The distance between the in focus specimen and the front lens of the objective • Depth of Field– (D.O.F) Depth of field in a microscope is the area in front of and behind the specimen that will be in acceptable focus • Field of View- (F.O.V) This is the area of the specimen in view down the eyepieces. It is dependant on the magnification and the Field number(F.N) of the eyepiece. F.N. of 22 indicates a F.O.V. diameter of 22mm when using a 1x objective • Convolusion – Areas above and below focal plane causing glare, distortion and blurriness
  35. 35. Orientation and Polarisation (end-on view) (end-on view) (end-on view) (end-on view)
  36. 36. Polarised light in Microscopy and Illumination Techniques Huygens’ Principle and Diffraction Pattern
  37. 37. Numerical Aperture (NA) and Working Distance (WD) sample WD WD WD Resolution
  38. 38. Let’s not get too involved here… Far away… NA 0.2 ABDFGRG BNHYUKF LGMNJDE GFKGLWL KLIIURRM Closer… NA 0.9 ABDFGRG BNHYUKF LGMNJDE GFKGLWL KLIIURRM
  39. 39. Jargon • Chromatic Aberration- When white light passes through a convex lens, the colours split and focus at different points causing colour fringing - Objectives have additional elements to overcome this problem • Spherical Aberration- When light passes through a material (glass/plastic) media • Apochromatic objectives are fully corrected and Achromatic objectives are corrected for red/blue • Plan objectives are designed, assuming a flat specimen, to provide a focused image across the whole field of view
  40. 40. Aberrations Chromatic Aberration Coma B A Spherical Aberration Astigmatism C On-Axis S T Off-Axis
  41. 41. Chromatic aberration
  42. 42. Objectives
  43. 43. Objective Lens Nomenclature Red = 4x Yellow = 10x Green = 20x Blue = 40 x
  44. 44. Objective Lens Types
  45. 45. Types of Objectives / Heads • No designation – Dry Objective / Non Oil • Infinity – High quality infinity corrected • Oil Objectives (Oil – OI) NA>0.9 must be oil • PLAN objectives (FN) Flat even focus across the Field of View (high quality)
  46. 46. High Mag. Objectives & Correction Collar • • • Corrects for Spherical Aberration Caused by variations in material thickness and type ITO stages Tokai vs Standard Different types of dish NUNC four well vs Falcon circular.. Rotation either increases or decreases the distance to first objective lens
  47. 47. Light-Gathering Power When we alter the light beam we affect the brightness. The more we do, the less light we have to push through and to see with, hence: Image Brightness: Numerical Aperture Magnification2 Correction Magnification Numerical Aperture F(trans) Plan Achromat 10x 0.25 6.25 Plan Apo 10x 0.45 20.2 Plan Fluorite 20x 0.50 6.25 Plan Apo 20x 0.75 14.0 Plan Achromat 40x 0.65 2.64 Plan Apo 40x (oil) 1.30 11.0 Plan Fluorite 60x 0.85 2.01 Plan Apo 60x (oil) 1.40 5.4 Plan Apo 100x (oil) 1.40 1.96 Plan Apo 100x (oil) 1.45 2.10
  48. 48. Eyepieces • • • • • • • Typically 10 x sometimes 15x Magnification These form an integral part of the magnification of the whole system Separate to any camera imaging Right eye is usually fixed and the left has a Dioptor adjuster Pupil distance adjustment for user comfort Head assembly and Prism split 50/50 prism = 50% to eyepieces 50 % to camera (basic head) Trinocular head has additional prism 100% to eyepieces or 20/80 split - usually only important for Darkfield and Fluorescence
  49. 49. COFFEE!
  50. 50. LUNCH & DINNER (Weather Dependant) • Dinner Tonight : – Choices • Sea Food • Combination • Beach • Italian LUNCH Menu choices on the board
  51. 51. Visualisation Techniques • The job of the light based equipment (Light, polariser, condenser, HMC etc) is to present the light for the objective to do its work • The objective is the key part and acts like our eye • Our human vision is triggered to see three dimensions and is well trained to interpret structures if they are illuminated more or less from one point. • We are limited by what we can see and the physics involved in the process • Remember that a compromise is just that…
  52. 52. Visualisation Techniques  Brightfield – Good for stained specimens  Darkfield – True colour against black background  Phase Contrast – Seeing live cells in plastic vessels  DIC – differential interference contrast Pseudo 3D of live cells in glass vessels only  HMC – Pseudo 3D of live cells in plastic vessels  Fluorescence – Sensitive method for localisation of chemical or physical changes (Tags)
  53. 53. Visualisation Techniques Brightfield Phase Contrast DIC Nomarski
  54. 54. Visualisation Techniques Brightfield Darkfield
  55. 55. What is Darkfield then.... • Stars in the sky.... – We cant see the stars in daylight on a clear day yet we know they are there... – We cant see dust the air in a room (most of the time!) • Yet if we shine a torch in a dark room we pick up the dust in our vision because they diffract and/or reflect the light • That is Darkfield or darkground illumination • Light is directed to the specimen in a way that no direct light enters the objective
  56. 56. Bright field IMAGE Objective Sample
  57. 57. Darkfield Cont. • Therefore we can see a particle or sample in front of a black background even when the particle itself is too small to be resolved or does not show an appropriate contrast under bright field IMAGE Objective Sample
  58. 58. Diagram of a dedicated Bright field – Dark field system
  59. 59. Oblique Illumination • The simplest way to achieve a contrast method that results in a relief-like image (2D/3D) is by using oblique illumination Direct Illumination Structures within a specimen can be identified and even though they are only two dimensionally displayed they look three dimensional. Oblique Illumination
  60. 60. Limits of Oblique illumination The contrast itself is produced by the complete thickness of the specimen and the resolution of the image is limited due to the oblique illumination To overcome the limitations of oblique contrast, Hoffman, Nomarski Differential Interference Contrast (DIC) and others are commonly used for high resolving images. The benefit of this method is that the relief like image is only contrasted at the focus area (depth of field). The user can optically section a thicker specimen by changing the focus level.
  61. 61. Polariser in Microscopy • Below the condenser, a circular polarizer is placed on the light exit port of the microscope. The rotation of this polarizer can control the effective width of the slit opening The part of the slit controlled by the polarizer registers on the bright area of the modulator. As the polarizer is rotated, contrast can be varied for best effect. A very narrow slit produces images that are very high in contrast.
  62. 62. HMC • Hoffman Modulation Contrast - Hoffman modulation contrast is an oblique illumination technique that enhances contrast in both stained and unstained specimens by detection of optical phase gradients.
  63. 63. Hoffman • The Hoffman Modulation Contrast system is designed to increase visibility and contrast in unstained and living material by detecting optical gradients (or slopes) and converting them into variations of light intensity. • Unlike the phase plate in phase contrast microscopy, the Hoffman modulator is designed not to alter the phase of light passing through any of the zones. • An optical amplitude spatial filter, termed a "modulator" by Hoffman, is inserted on the back focal plane of an achromat or planachromat objective (although higher correction can also be used). Light intensity passing through this system varies above and below an average value, which by definition, is then said to be modulated • When viewed under modulation contrast optics, transparent objects that are essentially invisible in ordinary brightfield microscopy take on an apparent threedimensional appearance dictated by phase gradients
  64. 64. Principles of light path in Hoffman
  65. 65. Visualisation Techniques ii Hoffman Modulation Contrast (HMC)
  66. 66. Condenser • Above the stage, a condenser with rotating turret is utilized to hold the remaining components of the Hoffman Modulation Contrast system. • The turret condenser has a brightfield opening with an aperture iris diaphragm for regular brightfield microscopy. • At each of the other turret openings, there is an off-center slit that is partially covered with a small rectangular polarizer. The size of the slit/polarizer combination is different for each objective of different magnification; hence the need for a turret arrangement.
  67. 67. HOFFMAN MODULATION CONTRAST i - HMC
  68. 68. Objective Condenser • The modulator has three zones that are depicted in Figure 2: a small, dark zone near the periphery of the back focal plane which transmits only one percent of light (areas marked "D" in Figure 2); a narrow gray zone which Three-dimensional transmits 15 percent (areas marked "G" in Figure 2); and the remaining appearance dictated the territory clear or transparent zone, covering most ofby phase at the back of the objective, which transmits 100 percent of the light (areas marked "B“) gradients
  69. 69. DIC / Nomarski • An excellent mechanism for rendering contrast in transparent specimens, differential interference contrast (DIC) microscopy is a beam-shearing interference system in which the reference beam is sheared by a minuscule amount, generally somewhat less than the diameter of an Airy disk. • The technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial frequencies present in the specimen. Those regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast. • As the gradient of optical path difference grows steeper, image contrast is dramatically increased.
  70. 70. Phase Contrast • Light that is travelling through part of a specimen and is not absorbed by amplitude objects will not produce a clearly visible image. The intensity remains the same, but the phase is changed compared to the light just travelling in the surrounding areas. This phase shift of about a quarter wavelength for a cultured cell is not visible to our eyes. Therefore, additional optical elements are needed to convert this difference into an intensity shift. These optical elements create a contrast where un-deviated and deviated light are ½ wavelength out of phase, which results in destructive interference. This means that details of the cell appear dark against a lighter background LIGHT Phase change
  71. 71. Diagram Principles of DIC / Nomarski
  72. 72. Visualisation Techniques Phase Contrast
  73. 73. Alignment of Visualisation Techniques - off axis aberration in phase contrast
  74. 74. Objective for Phase Microscopy
  75. 75. Diagram Principles of DIC / Nomarski
  76. 76. Visualisation Techniques Differential Interference Contrast (DIC)
  77. 77. Alignment of Visualisation Techniques - off axis aberration in phase contrast
  78. 78. HARDWARE MICROSCOPY Microscopes Stereo and Compound • Compound Microscopes use two lenses to focus light. 1st lens is the one nearest the object, 2nd lens is the one by the eye • Total magnification = objective x eye pieces (4x 10x = 40) through the eye pieces • Limit of resolution for light microscope is 1000x • 3 different types relevant to our work in IVF • Binocular - Two microscopes in one – offers observation of specimens at the natural conversion angle of our eyes. This makes the 3D topography visible
  79. 79. Light Microscope Schematic diagram of a light microscope outfitted for photomicrography And what’s the difference between this and the invert ?
  80. 80. Stereo Microscopes • Variety of Illumination options • Transmitted Brightfield, Darkfield, Oblique and Polarised Light • Fluorescence • Reflected through the optics or by separate source • Fibre optic goose neck or ring lights
  81. 81. Stereo zooms in IVF Typically either mounted in a class II Cabinet with the cabinet companies light source or as a stand alone unit on the microscopes light base. Uses Brightfield as standard.
  82. 82. Stereo Microscopes • 2 separate optical paths at an angle to each other • Brain merges the 2 images to give a 3D image . • Magnification range 4x- 400x • 2 principal types • Large working distances required
  83. 83. Greenhough Optics • 10 Degree Converging light path • Great depth of focus To see objects within a microscope in a similar way, both eyes have to each be provided with a separate light path to enable observation of the specimen at an angle similar to the one our eyes see from naturally
  84. 84. Galilean Optics •Parallel Optics •Allows build up of other accessories
  85. 85. Inverted Microscopes • Predominately used for looking at specimens in suspension- Live cells – ICSI • Requires Long working distance optics for manipulators • Whole variety of contrast techniques available • Environmental and temperature control on stages • Sample Manipulation/Injection • Magnifications 40x – 1000x (7000x with additions)
  86. 86. Inverted Scopes in IVF • • • • Typically only found with micro manipulators in IVF lab Featured here with the Integra base from RI Large body of evidence supporting the importance of anti vibration platforms combined with all manipulators Perfect set up and maintenance easy to achieve for both the Integra and the Microscope with the right training
  87. 87. Upright Microscopes • Samples on glass slides (Andrology) • Small Working distance • Variety of Contrast techniques used • Magnification range 10x – 1000x
  88. 88. The MicroscopeWhat is Important? • Location – Ideally in an area with few disturbances • Avoid direct lighting - not next to a window • Ensure access to working parts • Comfortable working area for using both eyepieces and manipulators • Adjustable chair for different eye heights • Isolate external vibration – important for manipulation and laser techniques • Consider an ergonomic head for the microscope
  89. 89. Imaging in the IVF Laboratory • The image from an optical microscope can be captured by normal light-sensitive cameras to generate a micrograph • Additional magnification can be achieved using imaging • Originally images were captured by photographic film but modern developments in CMOS and charge-coupled device (CCD) cameras allow the capture of digital images • Purely Digital microscopes are now available which just use a CCD camera to examine a sample, and the image is shown directly on a computer screen without the need for eyepieces
  90. 90. Beam Splitter / Prism
  91. 91. CCD Camera • • • Charged Couple Device - CCD Cameras are have microscope specific mountings on them which often feature a demag (x0.55/x0.7) to ensure the image is identical on the screen to what the scope sees Current trend is to mount the system on a PC for image manipulations or addition to a PIMS C-Mount Camera CCD Mounting (Demagnification)
  92. 92. Cleaning and Care • Never touch the lenses with your fingers. Your body produces an oil that smudges the glass • Oil can even etch the glass if left on too long • Never use paper or tissue to clean the glass of the microscope as they WILL scratch them • Use only approved Lens or cleaning tools to clean the glass* • *Avoid Alcohol on LCD displays as this may cause cracking*
  93. 93. Break
  94. 94. Manipulators START OF OUR PRACTICAL ICSI SESSIONS Please find yourself a system.
  95. 95. Controls Course Control x and y control 10 micron resolution 4mm x and y travel Stage Fine Control XYZ movement Sub-micron resolution 5mm z travel XYM
  96. 96. PL30 and MPH &Tool Holders Toolholder angle adjustment- 15 to 40 degrees. Vertical, axial and rotational movements MPH Micro Pipette Holder PL30
  97. 97. Tool holders • The tool holders provide the simplest and quickest method ever for setting-up micropipettes. The tool holders, when used with a special objective and spacer (supplied), allow the micropipettes to be set-up 14mm above the Petri dish and then rapidly lowered to the desired position, preventing micropipette damage. • The tool holders also have a special angular adjustment which allows the user to adjust the angle of the pipette with a single control without moving the tip of the pipette. Unique to RI, this feature makes it easy to ensure that the micropipette is slightly “toe-down” during sperm immobilisation and horizontal during sperm injection
  98. 98. Pipette Challenge Time to practice with the rigs Spend time with the optical path arrangements of each scope Understand what causes the optical changes you are seeing By the end of the day we will all have a time on the board for pipette set up BEST TIME 3.10 sec / Montana Diaz Diaz – Kings College London
  99. 99. What we are looking at is not always what it is and sometimes it is very well hidden! The art of good IVF microscopy is effective differentiation
  100. 100. Wednesday • Day 1 Morning DAY 1: 8.00 – 9.30 Reception The office will be open and able to receive trainees for Tea /Coffee reception (for early arrivals!) til 9.30am Please arrive each day in advance of the scheduled start time so we can start promptly Some attendees will have arranged for RI to pick them up from local accommodation - This will be carried out at this time Location: RI Head Office • 9.30 – 10.15 – Directors and UK Managers welcome Introductions and reception with a tour of the facilities and transfer down to the training Suite at Site B Location: Boardroom RI Head Office • 10.15 – 10.30 – Introduction and Training aims for the practical session Training aims and individual itinerary discussion Familiarisation with the format and availability of the range of equipment Location: Training Suite Site B. The rest of the sessions will take place here • 10.30 - 11.30 Session 1 Microscopy – Basics (Nikon, Olympus, Leica) Fundamentals of microscopy work and sample illumination • • 11.30 – 11.45 Short Break 11.45 – 12.15 Session 2 Microscopy – IVF Principles for Microscopy in the IVF application including proper set up of Condenser Illumination, Microscope and Optics • 12.15 – 12.45 Fundamentals of Micromanipulation Integra and Manipulation techniques • 12.45 – 13.45 Lunch – Off Site Location
  101. 101. Day 1 Afternoon • 13.45 – 15.45 – • • Micromanipulation Continued Practical session on the use of micromanipulators - including but not exclusively: • Pipette selection and set-up • Quick set up guide of Pipettes on stage • Understanding the TDU • Understanding the PL30 and MPH • Overview of the HUD and Heated stages – Limitations and Benefits of ITO surfaces • Trouble shooting for common errors in set up in an IVF Lab 15.45 – 16.00 16.00 – 16.45 Short Break System and Pipette set up challenge A fun recap on the practical learning's of the day’s study • 16.45 – 17.00 Close of Day Key learning's and Transfers to accommodation • 19.30 - Late Evening Events – Smart Casual Dinner and Drinks in a seasonally appropriate venue Details to be confirmed in the morning with the Students
  102. 102. END OF DAY 1 • • • • Transfer to Hotels with Rob & Martyn Confirmation of Hotel Pick up to be agreed with each of you. Please make sure you have our mobile numbers • Food and Drink provided by RI • 19.30 - Late Evening Events – Smart Casual
  103. 103. Tomorrow Morning • 8.30- 8.45 am PICK UP at Hotels for those who need it – Train transfers in the afternoon will be made direct from RI so please bring your cases etc – If you need to leave early for any reason please let us know and we will try to compress your individual training needs –Session starts at 9AM!
  104. 104. Day 2
  105. 105. Day 2 Morning • Thursday • • • Day 2 8.30 – 9.00 9.00 – 10.00 I NEED A FAVOUR... Hotel Transfers to RI Training Suite Meeting Start - Manipulator Mania A fun, practical challenge for two teams to enhance and develop both the understanding and use of the main assemblies and tools in micromanipulation learnt on day 1 and applied in these day’s sessions • 10.00 – 11.00 Introduction to Advanced Techniques Biopsy and IMSI use in IVF with the RI Saturn ACTIVE and RI IMSI • • 11.00 – 11.15 11.15 – 12.45 Short Break Manipulation and Biopsy I Instruction on using laser assisted techniques and software Open session and material available for a wide range of techniques either building on the previous days learning's or the start of new skills on more advances equipment • 12.45 – 13.15 Lunch – On/Off site catering
  106. 106. Day 2 Afternoon • 13.15 – 13.45 Manipulation and Biopsy II Further hands on practical time and practice on the latest available ICSI/PGD and IMSI equipment Throughout the sessions and during time when other equipment is taken up with other students instruction for use of the following environmental monitors will be available • 13.45 – 1400 • • Short Break and Sugar Cornwall’s best tradition to keep us going 14.00 – 15.15 Individual Development Session – The Final Session! Individual itinerary focus for students to cover any aspect of the two days as they and the trainer see fit There will be almost 1-1 coverage of each aspect of the two days learning at each work areas with a wealth of RI trainers available for support • 15.15 – 15.30 Close of Workshop and Thanks Transfer to accommodation or trains and departures • 15.30 – Late After Hours The RI UK team will be available in the training suite for a further hour for those staying on Anyone staying another night in Falmouth are welcome to join us again for an evening out
  107. 107. Integra Ti components
  108. 108. Syringes
  109. 109. Temperature variation with objectives 40.0 ITO Heated Stage with Objectives (4x 10x 20x) Bourn Hall 13/04/12 Ref 1 10x rotated 39.5 39.0 38.5 38.0 37.5 37.0 36.5 36.0 35.5 35.0 34.5 4 x in position 20x rotated
  110. 110. Parfocal Alignment
  111. 111. Alignment of Olympus Relief or Hoffman Modulation
  112. 112. Biopsy Session -
  113. 113. IMSI Session
  114. 114. Microscope Workshop In Association with Research Instruments Nikon, & Olympus Microscopes
  115. 115. Stay in touch rwatkins@research-instruments.com 01326370606 / 07795438952 Like us on Facebook Follow us on Twitter @RI_Limited Add us to your circle on Google+ Follow us on LinkedIn Subscribe on YouTube
  116. 116. Thank you!

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