Flow Cytometry in Microfluidics

Saransh Khandelwal
Technical Superintendent,
Department of Biomedical Engineering
IIT Hyderabad, 502284
PAGE 1
Flow
Cytometryin
Microfluidics

Content
Outline
Introduction
Understanding Flow Cytometry
Concept of Fluorescence in Flowcytometry
State-of-the-art application-based Research Paper
Conclusion and Future Aspects
PAGE 2

Introduction
Basic Concept
Parameters
Fluorescence involved in Flow
Cytometry
Flow Cytometry
PAGE 3

Flow Cytometry
 The measurement (-metry) of cells (cyto-) as they flow
past a detection system.
 First developed for single-cell analysis in the late 1960’s
 Technology to measure one cell/particle at a time in a
moving fluid stream
 By the late 1970s, instruments configured with two
LASERS [1]
 Measure and Quantify cells plus Cell Sorting
 Microfluidic Flow Cytometer: cells can be confined in
micrometer-sized chambers and channels which are
compatible to their intrinsic volume, thus enabling the
analysis of single cell with the minimized dilution or
buffer solution [1]
PAGE 4
Flow Cytometry
Paper Review Conclusion and Discussions Questions

Flow Cytometry
Fig.1 Basic schematic of Flow Cytometer
LASER
SAMPLE
FS-Detector
SS-Detector
To PC
Hydrodynamic
Focusing
Dichroic Mirror
Fig. 1: Basic schematic of Flow Cytometer [2]

Hydrodynamic Focusing
PAGE 6
Flow Cytometry
The sample is injected into an outer sheath stream. The
pressure differential and design of the flow cell creates
laminar flow where the sample core is focussed into a
stream of single particles within the outer sheath layer
Piezoelectric/Acoustic waves can also be used to improve
focusing.
Fig. 2: Hydrodynamic Focusing
Sheath
Stream
Sample
Stream

Scattering
PAGE 7
Flow Cytometry
Forward Scattering: Magnitude proportional to Size of the sample
Fig. 3: Forward scattering with different size of cells

Scattering
PAGE 8
Flow Cytometry
Side Scattering: Magnitude proportional to Granularity and
Structural Complexity
Fig. 4: Side scattering featuring the granularity of a cell

Parameters
PAGE 9
Flow Cytometry
• Small angle (0.5-5°)
• Corresponds to cell size
• Large angle (15-150°)
• Corresponds to internal granularity,
unevenness of the cell surface
Forward Scatter (FSC) Side Scatter (SSC)
Fig. 5: Voltage peak acquired after FSC and SSC

Parameters
PAGE 10
Flow Cytometry
Forward Scatter (FSC) Side Scatter (SSC)
Fig. 6: Scatter plot obtained after sampling through FSC and SSC setup

Dot plot
PAGE 11
Flow Cytometry
Fig. 7: Dot plotting from Forward and Side Scatter data

Scatter plot
PAGE 12
Flow Cytometry
Neutrophils
Monocytes
Lymphocytes

Fluorescence
PAGE 13
Flow Cytometry
Many fluorescent substances are used in
flow cytometry:
• Fluorochrome antibody conjugates
• Nucleic acid binding dyes
• Metabolic indicators
• Fluorescent protein
Most flow cytometers use filters and
detectors to measure ranges of light
that are associated with specific
fluorochromes
The advanced FCM have array of 16-28
detectors for capturing different range.
Fluorescent tagged
antibody or dye

Flow Cytometry
Stokes Shift
British Scientist Sir George G. Stokes who first
described fluorescence in 1852 and was
responsible for coining the term in honour of
the blue-white fluorescent mineral fluorite
(fluorspar)
He discovered the wavelength shift to longer
values in emission spectra that is known as
the Stokes Shift.
However, Plank’s Equation was derived in
1990.
Figure 8: Fluorophore Absorption and Emission Profiles [3]

Flow Cytometry
Dichromatic Mirror
 It is positioned at 450angle to both the incident illumination arriving
from the light source, as well as the optical axis of the microscope
 Light from the source passes through the defined bandpass excitation
filter, and is then reflected down into the objective by the DM to be
focused at the specimen plane
 Fluorescence emission is captured by the objective and directed back
through the DM, which in turn reflects most of the contaminating
excitation light back toward the light source
 Emission wavelengths passing through the DM are further purified by
the emission filter, before traveling to the eyepieces or the camera
image plane
Figure 9: Dichromatic Mirror[3]

Flow Cytometry
Filters
 Fluorophores that can absorb a quantity of
illumination, the percentage that will emit
secondary fluorescence is even lower.
 The fundamental problem in fluorescence
microscopy is to produce high-efficiency
illumination of the specimen, while
simultaneously capturing weak
fluorescence emission that is effectively
separated from the much more intense
illumination band.
 These conditions are satisfied in modern
fluorescence instruments by a combination
of filters that coordinate excitation and
emission requirements.
Fig. 10: Filters characteristics [3]

Flow Cytometry
Advancements and Current Techniques
 Usually, sample pumping, focusing and sorting are achieved on a microfluidic
chip.
 Due to the extremely small size of the microchip, less volume of sample and
reagent is usually required in the microfluidic flow cytometer resulting in
reducing the cost significantly
 Cells can be assayed one at a time at speeds of up to 100,000 cells per second
 High-end cytometers can measure up to 30 parameters (28 colours + FSC/SSC)
 Cell sorting speeds up to 25,000 per second

Flow Cytometry
Applications and state of the art equipment's
 Cell sorting – cells are not destroyed
 Pumping, Sampling, Dispensing Sequential Loading etc
 POC diagnostic tests
 Haematology, Oncology, Immunology
 Blood Banking
• LSR II – 5 laser
• LSR Fortessa 4 laser
• 2 x LSR Fortessa X20 4 laser
• LSR Fortessa X50 – 5 laser
• 3 x Attune NxT – 4 laser
• FACSAria II
• FACSAria III
• 2 x Aria Fusion
• Sony MA900

Flow Cytometry
Limitations and Future Aspects
With the rapid development of electronics and computer science, the data processing is much
easier to be miniaturized and integrated into the systems. However, compared to the conventional
flow cytometers, the microfluidic flow cytometers also exhibit some limitations
 Lower transfer rates
 Integration and simplification of many functional components on a microchip still is a challenge
 The standardization of advanced microfluidic flow cytometers in clinical and research laboratories
is the key to expand these technologies rapidly
The sensitivity and throughput of such systems need to be further improved. Besides, parallel
detection and processing is used to improve the throughput. [4]
 A highly parallel acoustic flow cytometer that used an acoustic standing wave to focus particles
into 16 parallel analysis points.
 Smartphone-Based Analysis of single RBCs [5]

Flow Cytometry
Paper Review
Conclusion and Discussions Questions
Paper Review
 Biosensors and
Bioelectronics
 Impact Factor: 12.545
 Volume 222, 15 February
2023, 114916

Flow Cytometry
Paper Review
Introduction
 Conventional immunophenotyping techniques such as flow cytometry or fluorescence microscopy require
immunolabeling of cells, expensive and complex instrumentation, skilled operators, and are therefore
incompatible with field deployment and automated cell manufacturing systems.
 In this work, an autonomous microchip that can electronically quantify the immunophenotypical
composition of a cell suspension.
 Microchip identifies different cell subtypes by capturing each in different microfluidic chambers
functionalized against the markers of the target populations.
 All on-chip activity is electronically monitored by an integrated sensor network, which informs an
algorithm determining subpopulation fractions from chip-wide immunocapture statistics in real time.
 Microchip analyzed a mixture of unlabelled CD4+ and CD8+ T cell sub-populations and validated the
results against flow cytometry measurements.

Flow Cytometry
Paper Review
Theory
 Multicolour flow cytometry has thus far served as the gold standard for immunophenotype
measurements.
 Cells are first pre-labeled with specific antibodies that have been conjugated with fluorochromes and
then interrogated under laser illumination.
 Seeking to create low-cost, portable cell immunophenotyping assays, researchers have
developed microchip-based technologies.
 Typically, such systems drive a cell suspension through an antibody-functionalized microchip
which in turn screens the cell population constituents using immunoaffinity as the discriminatory
mechanism.
 To achieve this capability, this work combines microfluidics, integrated on-chip sensors, computation and
real time feedback control.

Flow Cytometry
Paper Review
Design of
Experiment
 Microchip utilizes a multi-
chamber immunocapture
scheme
 Separates cell
subpopulations by capturing
them based on their surface
markers
 An integrated electrical
sensor network to quantify
the capture statistics
Fig. 11: A schematic illustrating the method for electronic immune cell analysis [6]

Flow Cytometry
Paper Review
Fabrication
Fig. 12: A photograph of the fabricated microchip showing the microfluidic features filled with red dye for visualization and the electrical sensor network
formed by micropatterned gold electrodes. Insets show a close-up microscope image of (top) one of the coded electrical sensors on the device along with
the inter-chamber microfluidic passage it monitors and (bottom) a group of micropillars within the capture chamber that cells interact with as they flow
through the device. [6]
 15 μm thick negative
photoresist SU-8 was spin
coated on Si wafer
 Design Pattern was
transferred
 a mixture of PDMS
elastomer and its crosslinker
was prepared at a 10:1 ratio
(by weight) and poured onto
the mold.
 Cured PDMS was peeled off

Flow Cytometry
Paper Review
Design of
Experiment
 The readout produced by
the sensor network is
analysed by custom-built
deep learning algorithms.
 Algorithms also inform a
runtime feedback control
algorithm that continually
monitors the cell flow
dynamics
 Maintain optimal cell flow
conditions for
immunocapture
Fig. 13: A schematic showing all of the components of the developed autonomous microchip-based immunoassay
system and their interaction [6]
 The readout produced by
the sensor network is
analysed by custom-built
deep learning algorithms.
 Algorithms also inform a
runtime feedback control
algorithm that continually
monitors the cell flow
dynamics
 Maintain optimal cell flow
conditions for
immunocapture

Flow Cytometry
Paper Review
Results
 The complete system
consisting of the developed
hardware and software by
processing heterogenous
samples of mixed T cell
populations.
 The first chamber would
capture exclusively CD4+ T
cells, the second chamber
would capture CD8+ T cells
 To confirm that both
capture chambers were
capturing their intended
target cell populations,
imaging was done with
fluorescence microscopy. Fig. 14: Schematic of the device showing the layout of capture chambers. Insets show images of CD4+ (left) and
CD8+ (right) cells labeled fluorescently after they were captured on the device [6]

Flow Cytometry
Paper Review
Results
Fig. 15: A plot showing the CD4+ and CD8+ T cell subpopulation frequencies as reported by the system (color-filled bars) vs the nominal mix ratio determined
by hemacytometer (unfilled bars). Consecutive image gives the difference between the subpopulation counts determined by flow cytometry vs. our
microchip-based immunoassay [6]

Flow Cytometry Paper Review
Conclusion and Discussions
Questions
Conclusion and Discussions
 The percentage of CD4+CD8− T cells, CD4−CD8+ T cells, and CD4−CD8−expressors were 48.3%,
46.8%, and 4.1% respectively as per flow cytometry results.
 These results were in good agreement with those from the designed immunoanalysis system,
which reported 49.3%, 43.8% and 6.9% as the frequencies of aforementioned
immunophenotypes.
 Considering the flow cytometry data as the ground truth, these results amounted to an average of
≤6%, effectively representing an accuracy of >94% for the designed system.
 Conclusions are supported by the fact that the used CD4+ T cell and CD8+ T cell samples had
manufacturer-reported purities of 94% and 93%, respectively.
 A proof-of-concept implementation that targets two surface markers, the scalable nature of our
immunocapture scheme enables a straightforward application of our approach to target a
multitude of surface markers by simply adding chambers to the design and functionalizing them
accordingly.
 The measurement throughput can be further increased by revising the fluidic design to lower the
hydraulic resistance.

Flow Cytometry Paper Review Conclusion and Discussions
Questions
Questions…??

References
PAGE 30
1. New advances in microfluidic flow cytometry, Yanli Gong et.al., Wiley Analytical Science, Electrophoresis, Volume 40, Issue
8, P.P. 1212-1229, 2019
2. Flow Cytometry: Principles and Clinical Applications in Hematology, Brown M and Wittwer C. et.al. ,Clin Chem 46(8) P.P.
1221-29, 2000
3. https://zeiss-campus.magnet.fsu.edu/articles/basics/fluorescence.html
4. Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow
Cytometry, Kalb, D. M., Fencl et.al. Anal. Chem., 89, 9967-9975, 2017
5. A smartphone-based optical platform for colorimetric analysis of microfluidic device, Kim, S.C.; Jalal, U.M.; Im, S.B.; Ko, S.;
Shim, J.S.. Sens. Actuators B Chem, 239, 52–59, 2017
6. An autonomous microchip for real-time, label-free immune cell analysis, A.K.M. Arifuzzman et.al. Biosensors and
Bioelectronics Volume 222, 114916, 2023

Flow Cytometry in Microfluidics

Recommended

Recommended

More Related Content

Similar to Flow Cytometry in Microfluidics

Similar to Flow Cytometry in Microfluidics (20)

Recently uploaded

Recently uploaded (20)

Flow Cytometry in Microfluidics

Editor's Notes