Replica of microscope by Van Leeuwenhoek ~1660 Van Leeuwenhoek's interest in microscopes and a familiarity with glass processing led to one of the most significant, and simultaneously well-hidden, technical insights in the history of science. By placing the middle of a small rod of soda lime glass in a hot flame, Van Leeuwenhoek could pull the hot section apart like taffy to create two long whiskers of glass. By then reinserting the end of one whisker into the flame, he could create a very small, high-quality glass sphere. These spheres became the lenses of his microscopes, with the smallest spheres providing the highest magnifications. An experienced businessman, Leeuwenhoek realized that if his simple method for creating the critically important lens was revealed, the scientific community of his time would likely disregard or even forget his role in microscopy. He therefore allowed others to believe that he was laboriously spending most of his nights and free time grinding increasingly tiny lenses to use in microscopes, even though this belief conflicted both with his construction of hundreds of microscopes and his habit of building a new microscope whenever he chanced upon an interesting specimen that he wanted to preserve. Van Leeuwenhoek used samples and measurements to estimate numbers of microorganisms in units of water.   Van Leeuwenhoek made good use of the huge lead provided by his method. He studied a broad range of microscopic phenomena, and shared the resulting observations freely with groups such as the English Royal Society .  Such work firmly established his place in history as one of the first and most important explorers of the microscopic world.
Microscopic Section through one year old ash tree ( Fraxinus ) wood, drawing made by Van Leeuwenhoek.
Fluorescence micrographs (low and high magnification) of a neuron co-transfected with mitochondrially-targeted yellow fluorescent protein and cytoplasmic cyan fluorescent protein.
Image of bioluminescent red tide event of 2005 at a beach in Carlsbad California
Progress in transfection technology was relatively slow until the advent of molecular biology techniques for cloning plasmid DNA. These techniques provided the means to prepare and manipulate DNA sequences and the ability to prepare virtually unlimited amounts of relatively pure DNA for transfection experiments. Cloned sequences could also be used to generate RNA in vitro with phage RNA polymerase using DNA templates with the corresponding polymerase promoter (3). As the ability to prepare DNA and RNA for transfection became easier, additional methods, such as electroporation and liposome-mediated transfer, were developed to enable more efficient transfer of the nucleic acids to a broad range of cultured mammalian cells (4,5). The development of reporter gene systems and selection methods for stable gene expression of transferred DNA greatly expanded the applications for gene transfer technology (Figure 1.1). In 1982, Gorman et al. initiated the reporter gene concept with the bacterial chloramphenicol acetyltransferase (CAT) gene and associated CAT assay system (6). Using a reporter gene that is not endogenous to the cell, coupled with a sensitive assay system for that gene product, allows investigators to clone regulatory sequences of interest upstream of the reporter gene to study expression of the reporter gene under various conditions. This technology, together with the availability of transfection reagents, provides the foundation for studying promoter and enhancer sequences, trans-acting proteins such as transcription factors, mRNA processing, protein/ protein interactions, translation, and recombination events (7). Since the introduction of the CAT gene and assay system several other reporter systems have been developed for various in vitro and in vivo applications including luciferase, b-galactosidase, alkaline phosphatase and green fluorescent protein (7). See Chapter 6 for detailed descriptions of Promega’s luciferase, CAT and b-galactosidase reporter vectors and assay systems. Integration of DNA into the chromosome, or stable episomal maintenance, of reporter genes and other genes occurs with a relatively low frequency. The ability to select for these cells is made possible using genes that encode resistance to a lethal drug. An example of such a combination is the marker gene for neomycin phosphotransferase with the drug Geneticin® (8). Individual cells that survive the drug treatment expand into clonal groups that can be individually selected, propagated and analyzed. Today the study of gene regulation, the analysis of the expression and function of proteins within mammalian cells, the generation of transgenic organisms and in vivo/ex vivo gene therapy strategies are all made possible by the availability of of gene transfer technologies, nucleic acid molecular biology and reporter gene systems.
The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins.
Video 2 (2.9 MB) Mitochondrial dynamics in axons of hippocampal pyramidal neurons: co-transfection with the control, pIRES-GFP Hippocampal neurons were transfected with pDsRed1Mito + pIRES-GFP at 3 DIV. Time lapsed images were obtained of living neurons at 6 DIV capturing an image every 6 seconds for a total of 5 min (total images = 50). The video is shown at 30 x real time.
P hotoconversion of a PS-CFP2 fluorescent protein
Direct microinjection into cultured cells or nuclei is an effective, although laborious technique to deliver nucleic acids into cells. This method has been used to transfer DNA into embryonic stem cells that are used to produce transgenic organisms. However, this technique is not appropriate for studies that require a large number of transfected cells. Electroporation was first reported for gene transfer studies in 1982. This technique is often used for cell types such as plant protoplasts that are particularly recalcitrant to milder methods of gene transfer. The mechanism for entry into the cell is based upon perturbation of the cell membrane by an electrical pulse, which forms pores that allow the passage of nucleic acids into the cell. The technique requires fine-tuning and optimization for duration and strength of the pulse for each type of cell used. A critical balance must be achieved between conditions that allow efficient delivery and conditions that kill cells. Another physical method of gene delivery is biolistic particle delivery . This method relies upon high velocity delivery of nucleic acids on microprojectiles to recipient cells. This method has been successfully employed to deliver nucleic acid to cultured cells, as well as to cells in vivo.
Electroporation was first reported for gene transfer studies in 1982. This technique is often used for cell types such as plant protoplasts that are particularly recalcitrant to milder methods of gene transfer. The mechanism for entry into the cell is based upon perturbation of the cell membrane by an electrical pulse, which forms pores that allow the passage of nucleic acids into the cell. The technique requires fine-tuning and optimization for duration and strength of the pulse for each type of cell used. A critical balance must be achieved between conditions that allow efficient delivery and conditions that kill cells. Another physical method of gene delivery is biolistic particle delivery . This method relies upon high velocity delivery of nucleic acids on microprojectiles to recipient cells. This method has been successfully employed to deliver nucleic acid to cultured cells, as well as to cells in vivo.
Real-time visualization of SCE of TOTO-1 labeled pDsRed2-C1 and subsequent expression of DsRed (A) Overlays of the IR and fluorescence image are shown at the indicated time points (in s) after starting the pulse. The pipette tip was pulled back shortly after pulse application. (B) 24 h after SCE, weak green fluorescence of the TOTO-1 labeled plasmid was visible in the soma. (B ’ ) The same soma in B, visualized by the red fluorescence of DsRed expression. (B ” ) Overlay of B and B ’ . (C) Unlabeled pDsRed2-C1 electroporation leads to strong expression of DsRed. Scalebar in A, 10 mm, in Bƒ, 20 mm, in C, 100 mm.
Transfection Technologies Many transfection techniques have been developed. Desirable features include high efficiency transfer of nucleic acid to the appropriate cellular organelle (for example, DNA into the nucleus), minimal intrusion or interference with normal cell physiology, low toxicity, ease of use, reproducibility, successful generation of stable transfectants, and in vivo efficacy. The techniques developed for gene transfer can be broadly classified as either chemical reagents or physical methods. Chemical Reagents DEAE-dextran was one of the first chemical reagents used for transfer of nucleic acids into cultured mammalian cells (1,9). The ProFection® Mammalian Transfection System-DEAE-Dextran provides reagents for this transfection technique (see Chapter 4 for further information). DEAE-dextran is a cationic polymer that associates with negatively charged nucleic acids. An excess of positive charge, contributed by the polymer in the DNA/polymer complex allows the complex to come into closer association with the negatively charged cell membrane. Uptake of the complex is presumably by of nucleic acids into cells for transient expression; that is, for short-term expression studies of a few days in duration. However, this technique is not generally useful for stable transfection studies that rely upon integration of the transferred DNA into the chromosome (10). Other synthetic cationic polymers have been used for the transfer of DNA into cells, including polybrene (11), polyethyleneimine (12) and dendrimers (13,14). Calcium phosphate co-precipitation became a popular transfection technique following the systematic examination of this method by Graham and van der Eb in the now-classic paper published in 1972 (2). Their study examined the effect of different cations, cationic and phosphate concentrations, and pH on the parameters of transfection. Calcium phosphate co-precipitation is widely used because the components are easily available and reasonable in price, the protocol is easy to use and many different types of cultured cells can be transfected. This method is routinely used for both transient and stable transfection of a variety of cell types. The protocol involves mixing DNA with calcium chloride, adding this in a controlled manner to a buffered saline/phosphate solution and allowing the mixture to incubate at room temperature. This step generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken-up by the cells via endocytosis or phagocytosis. The calcium phosphate also appears to provide protection against intracellular and serum nucleases (15). Promega’s ProFection® Mammalian Transfection System-Calcium Phosphate provides reagents for this transfection technique (see Chapter 4 for further information). By 1980, artificial liposomes were being used to deliver DNA into cells (5). The next advancement in liposomal vehicles was the development of synthetic cationic lipids by Felgner and colleagues (16). Liposome-mediated delivery offers advantages such as relatively high efficiency of gene transfer, ability to transfect certain cell types that are intransigent to calcium phosphate or DEAE-dextran, successful delivery of DNA of all sizes from oligonucleotides to yeast artificial chromosomes (16-20), delivery of RNA (21), and delivery of protein (22). Cells transfected by liposome techniques can be used for transient and for longer term experiments that rely upon integration of the DNA into the chromosome or episomal maintenance. Unlike the DEAE-dextran or calcium phosphate chemical methods, liposome-mediated nucleic acid delivery can be used for in vivo transfer of DNA and RNA to animals and humans (23). A lipid with overall net positive charge at physiological pH is the most common synthetic lipid component of liposomes developed for gene delivery (Figure 1.3). Often the cationic lipid is mixed with a neutral lipid such as L-dioleoyl phosphatidylethanolamine (DOPE) (Figure 1.4). The cationic portion of the lipid molecule associates with the negatively charged nucleic acids, resulting in compaction of the nucleic acid in a liposome/nucleic acid complex. For cultured cells, an overall net positive charge of the liposome/nucleic acid complex generally results in higher transfer efficiencies, presumably because this allows closer association of the complex with the negatively charged cell membrane. Following endocytosis, the complexes appear in the endosomes, and later in the nucleus. It is unclear how the nucleic acids are released from the endosomes and traverse the nuclear membrane. DOPE is considered a “fusogenic” lipid (24) and it is thought that its role may be to release these complexes from the endosomes, as well as to facilitate fusion of the outer cell membrane with the liposome/nucleic acid complexes. Promega provides a variety of transfection reagents that use cationic lipids for the delivery of nucleic acids to eukaryotic cells. These include TransFast™ Transfection Reagent, the Tfx™ Reagents and Transfectam® Reagent. See Chapter 3 for more information on the use of these reagents. endocytosis. This method is successful for delivery
Flow chart of the main procedures of Ca2+ phosphate transfection protocol
Formation of optimal DNA/Ca2+-phosphate precipitate and subsequent dissolution to stop transfection (a) Continuous vortexing when mixing DNA with Ca2+ and phosphate buffer results in large clusters of precipitate. The precipitate was examined after 1 h incubation. (b, c) Formation of optimal DNA/Ca2+-phosphate precipitate achieved by gently vortexing the DNA/Ca2+ solution with phosphate buffer. Image taken after 1 h incubation. (d) Dissolving precipitate with slightly acidic transfection medium preequilibrated in a 10% CO2 incubator in order to reduce its toxicity to neurons. Scale bar, 50 mm.
Transmission electron micrograph of multiple bacteriophages attached to a bacterial cell wall Virus entry requires sequential interaction between specific viral membrane glycoproteins and cellular receptors. Much of the recent work elucidating these receptors and the viral glycoproteins interacting with them has been carried out in the laboratories of P. Spear (Northwestern University), and G. Cohen and R. Eisenberg of the University of Pennsylvania. Upon entry the nucleocapsid is transported to the nuclear pores, where viral DNA is released into the nucleus. The viral genome is accompanied by the a-TIF protein which functions in enhancing immediate early viral transcription via cellular transcription factors. The virion-associated host shutoff protein ( vhs --UL41) appears to remain in the cytoplasm where it causes the disaggregation of polyribosomes and degradation of cellular and viral RNA.
Rat neocortical neurons infected with recombinant Sindbis virus-enhanced green fluorescent protein in vivo imaged in fixed tissue sections
Transfection of hippocampal neurons in low-density microisland cultures facilitates electrophysiological analysis
Hippocalcin translocation in dendritic shaft was assessed by FRET between HPCA1-14-CFP and HPCA-YFP. A demonstrates a part of dendritic tree where hippocalcin translocation was observed as a result of network bursting. Hippocalcin fluorescence changes and FRET efficiency in ROIs shown in red and green in A are demonstrated in B and C. E, F and G. Translocation changes follows changes in FRET indicating that local plasma membrane hippocalcin association is a reason for the translocation. H. An electrical activity simultaneously recorded in the neuron in a cell-attached configuration. D. Averaged changes (over 16 pairs of red and neighboring green ROIs) in hippocalcin fluorescence and FRET efficiency. I. The same set of sites engaged in hippocalcin signaling possibly indicating distribution of hippocalcin membranous targets.
A real-time movie of confocal real color images and corresponding pseudocolored ratio images showing Ca2+ waves inside cells, which were evoked by histamine, in HeLa cells expressing YC3.60(Yellow Cameleon). The images were taken at video rate (30 Hz) using a color 3CCD camera for simultaneous acquisition of CFP (cyan-emitting mutant of GFP) and YFP (yellow-emitting mutant of GFP) images. To improve z axis resolution, a spinning disk confocal unit was placed in front of the camera. Ten micromolar histamine was added to the recording medium to activate receptor-evoked Ca2+ release from endoplasmic reticulum.
Design and plasma membrane expression of VSFP2s A: A pair of CFP (donor) and YFP (acceptor) is attached to the 4-transmembrane-voltage-sensing domain (VSD) of Ci-VSP. B, C: Confocal fluorescence (B) and transmission images (C) of PC12 cells transfected with VSFP2D. Note the targeting of the fluorescent protein to the plasma membrane. Scale bar is 30 μm.
1. Bogomoletz Institute of Physiology Kiev Ukraine Dr. Pavel Belan <ul><li>Fluorescent proteins in current biology </li></ul>
2. Lecture Plan <ul><li>Development of optical microscopy </li></ul><ul><li>Basics of fluorescence </li></ul><ul><li>Basics of molecular biology </li></ul><ul><li>Green Fluorescent Protein ( GFP) in the current biology </li></ul><ul><li>Gene delivery techniques </li></ul>
3. Replica of microscope by Van Leeuwenhoek
4. Microscopic section through one year old ash tree
8. <ul><li>The rapid advances made in developing live-cell imaging microscopes based upon low light level electron multiplying CCD cameras and different confocal systems, allows imaging fluorescent probes and optical highlighters over the entire visible and near-infrared spectral regions with near real-time precision and submicron space resolution. </li></ul>
9. DIC image of cultured hippocampal neurons
10. Fluorescent image of double labeled cultured hippocampal neurons Biochimica et Biophysica Acta 1802(1):143-50. 2010
11. Basic principles of fluorescence
12. Marking proteins by fluorescent tags Fluorescent tag Protein of interest
13. Problems with fluorescent tags Cell impermeability unspecificity
14. How can we specifically tag proteins by fluorescent markers ? How to do that in living cells ?
15. Gene expression
16. Reporter gene expression Gene under study Reporter fluorescent protein Protein under study
17. Himeric gene expression FP gene Gene under study Himeric fluorescent protein Gene under study
18. Plasmid construction
19. &quot;Bioluminescent dinoflagellates (Lingulodinium polyedrum) lighting a breaking wave at midnight. The blue light is a result of a luciferase enzyme (like firefly luciferase, but the enzyme in L. polyedrum shares no similarity with that of the firefly enzyme). Under the right conditions, the dinoflagellates become so numerous that the water takes on a muddy reddish color (hence the name &quot;Red Tide&quot;). Image of bioluminescent tide event at a beach in Carlsbad California http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
20. Aequorea victoria http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
21. Studies of protein expression using GFP как главный репортерный белок GFP It is possible to report gene expression in single living cells
22. First study describing Green Fluorescent Protein (GFP) as a protein expression marker Science 1994:Vol. 263. no. 5148, pp. 802 - 805 DOI: 10.1126/science.8303295
23. Olympus website
24. Diversity of Fluorescent proteins http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
25. Examples of Labeling by Different Fluorescent Proteins http://www.conncoll.edu/ccacad/zimmer/GFP
26. Pyramidal neurons of « rainbow » rats
27. J Biol Chem. 2011 Mar 30. [Epub ahead of print] Mitochondrial dynamics in axons of hippocampal pyramidal neurons
28. llustrated is the photoconversion of a PS-CFP2 fluorescent protein fusion product with human beta -actin using a 405-nanometer diode laser for imaging and conversion, as well as the argon-ion 488-nanometer spectral line for imaging and tracking of the photoconverted protein. P hotoconversion of a PS-CFP2 fluorescent protein as a way to study protein polymers
29. Examples of in vivo FP expression
30. How can we introduce plasmids that are large circular DNA molecules into the cells ? Transfection and infection as methods to introduce gene constructs
31. Direct microinjection into cultured cells or nuclei Physical Transfection Methods
32. Electroporation B iolistic particle delivery Physical Transfection Methods
33. Single Cell E lectroporation (SCE) Physical Transfection Methods
34. Various transfection technologies based on chemical reagents Chemical Transfection Methods
35. Flowchart of the main procedures of Ca 2+ phosphate transfection protocol Chemical Transfection Methods
36. Formation of optimal DNA/Ca2+-phosphate precipitate and subsequent dissolution to stop transfection Chemical Transfection Methods
37. Viral infection as a way to deliver genetic materials into the cells
38. Virus infection technique
39. Rat neocortical neurons infected with recombinant Sindbis virus-enhanced green fluorescent protein in vivo A ) Image of the injection site (arrow) and surrounding area (postnatal day [P] 11). ( B ) Higher magnification images (P 14). Left , layer 2/3 pyramidal neurons. Middle , layer 5 interneuron. Right , layer 5 pyramidal neurons. ( C ) 2-photon laser scanning microscopy image of a layer 2 pyramidal neuron in a section from a mouse (P 36). Lern. Mem. 7, pp. 433-441, 2000 .
40. (a1) Overlay of fluorescent and phase-contrast images illustrating a pair of transfected (green) and nontransfected neurons. (a2) Double whole-cell patch-clamp recording of the pair of neurons shown in (a1). Upper trace shows presynaptic current and lower trace the corresponding postsynaptic current. (b) A pair of neurons both transfected with EGFP. Transfection of hippocampal neurons in low-density microisland cultures
41. Fast protein translocation study by means of GFP Eur. J. Neurosci. (2010) 32:347
42. <ul><li>Conclusions </li></ul><ul><li>The battery of fluorescent proteins, tools for their genes delivery and imaging tools enables the cell biologists to readily monitor protein dynamics in living cells, continuing to provide numerous new insights into the behavior of proteins, organelles, and cells. In so doing, these remarkable probes have ushered in a new era of cell biology in which both steady-state and kinetic microscopy techniques can be used to decipher pathways and mechanisms of biological processes. </li></ul>
43. <ul><li>Basics of FRET </li></ul><ul><li>Combining FRET with molecular biology techniques </li></ul><ul><li>Introduction to optogenetics </li></ul>Lecture Plan
44. Can we study inter- and intramolecular interection in living cells using GFPs? Do we have enough spatial resolution with our conventional microscope systems ?
45. Optical microscopy spacial resolution <ul><li>Compound optical microscopes are limited in their ability to resolve fine details by the properties of light. Optical microscope resolution depends upon the microscope objective and refraction properties of medium between the object and objective lens altogether characterized by the objective numerical aperture, A N . At the certain wavelengths of light (λ), the maximal resolution ( d ) is given by: </li></ul><ul><li>In case of λ = 550 nm (green light) and air as the medium, (the highest practical A N is 0.95), the maximal resolution is about 0.3 micrometers. At the same time due to aberrations, even the best optical microscope is limited to a resolution of around 0.5 micrometers. </li></ul>
50. The efficiency of FRET, E FRET , which is defined as probability of the occurrence of energy transfer per donor excitation event, is a steep function of the distance between the fluorophores, r, as given by the following equation: E FRET where Ro is the Forster distance http://www.olympusfluoview.com/applications/fretintro.html
54. Experimental Setup for Ratiometric FRET Imaging DualView CCD camera Inverted Microscope
55. The MAG Biosystems™ Dual-View utilizes a single beamsplitter to split the incident beam from the microscope into two independent beams. One beam contains all the emission reflected off of the beamsplitter; the other contains all the emission transmitted through the beamsplitter. Each of these emission channels is projected onto half of the CCD array at exactly the same point in time. Simultaneous multichannel imaging is essential to achieve quantitative emission ratiometric analysis.
57. Cameleon – FRET based Ca 2+ probe Cameleons have been devised by Roger Tsien and others. They are based on fluorescence resonance energy transfer (FRET) between two fluorescent molecules that are linked by a short stretch of calmodulin, a protein that changes its shape in the presence of calcium. In the absence of calcium the two fluorescent proteins are well separated. These calcium sensors are called Cameleons because they change color and have a long tongue (calmodulin) that retracts and extends in and out of its mouth when it binds and releases calcium.
58. Cameleon – основанный на FRET Ca 2+ зонд Tsien’s Lab
59. Design and plasma membrane expression of VSFP2s PLoS ONE , doi:10.1371/journal.pone.0000440
60. Protein kinase G activity measured by FRET Tsien’s Lab
61. FRET summary movie
62. <ul><li>Conclusions </li></ul><ul><li>Fluorescent proteins together with current biophysical and molecular biology approaches are the powerful tool allowing to study practically any type of inter- and intramolecular interection in living functional cells and thereby they can be used to decipher pathways and mechanisms of biological molecular signaling processes. </li></ul>
63. <ul><li>Can we not only study molecular signaling processes in living cells using optically active proteins but also control these processes in cell specific manner ? </li></ul>
64. Optogenetics Nature web site
65. Conclusions <ul><li>Optogenetics is a new experimental methodology based upon the combination of genetic and optical methods to control specific mainly electrical events in targeted cells of living tissue . </li></ul><ul><li>Optogenetics allows to almost instantaneously switch on and off certain neuronal groups in order to study their functions and relationships with other cells of animal body. </li></ul><ul><li>Optogenetics also allows to correct pathological changes of signaling processes being a prerequisite for treatment of many neurodegenerative disorders . </li></ul>