Lecture 1 – introduction/ Single cell technologies, modalities
Immunotherapy = type of treatment in which the immune system is modulated (enhanced or suppressed). There is
emphasis on cellular heterogeneity, particularly in solid tumors, where a small fraction of molecularly distinct cells such
as cancer stem cells contribute to tumor progression and therapeutic resistance.
- Single cell techniques allow you to study rare cells which is important in tumors for example to understand
their biology.
o Single cell applications
▪ Immune monitoring and immunophenotyping: identification and tracking of the immune cell
populations involved in tumor responses
▪ Cell migration and tumor killing: tracking how immune cells move and interact with tumors
▪ Structural and functional characterization: using advanced imaging techniques to understand
cellular and subcellular dynamics in real-time
- Examples of technologies:
o Cytometry and sorting which includes high-dimensional flow cytometry and live-cell sorting
o Microscopy: Advanced imaging techniques like confocal microscopy, super-resolution imaging (light-
sheet microscopy), and live-cell imaging.
o An emerging method is multidimensional data analysis which is key for handling large datasets
generated from cytometry and microscopy
The life sciences are now moving towards the trans-omics field in which we can integrate all the information of each
level
Cytometry = a technology that measures properties of single cells. It involves the quantitative analysis of physical and
chemical properties of cells or other biological particles, typically at the single-cell level.
- Parameters it measures:
o The cell surface markers are measured by fluorescence
o Light scatter measures the cell size and shape
o Using fluorescent dyes and antibodies it can also measure intracellular molecules
o And other properties of cell
- It provides correlated data that links different population profiles.
Flow cytometer = technology used to measure properties of single cells. It is a system that measures and analyzes
signals resulting from flowing particles in a liquid stream through a beam of light. It does not always deal with cells but
also chromosomes, vesicles, latex beds, or any particles that can be suspended in a fluid.
- The work comes from the Greek “cyto” (Cell) and “metry” (measurements). Early cytometers, known as FACS
(fluorescence activated cell sorters) were designed to sort cells based on fluorescence. They were the first ever
instruments made from the principle of separating cells without measuring what type of cells they really are.
- What can we do with a flow cytometer:
o Count cells
o Distinguish between biological and non-biological
o Separates live from dead
o Evaluate 105 to 107 particles in less than a minute
, o You can measure particle-scatter, autofluorescence, or fluorescence associated to other reagents
(antibodies and non-antibodies)
o Sort single particles/cells for subsequent analysis
o But also, more advanced capabilities:
▪ Obtain images
▪ Measure rare metals
▪ Measure the entire spectrum of light
Now, there are more technologies that use the principle to analyze one cell at a time but looking at different particles:
- Drop-seq dingle cell analysis
This is a platform to profile thousands of cells by encapsulating them into
individual droplets. Uniquely barcoded mRNA capture microparticles and
cells are co-confined through a microfluidic device within the droplets
where they undergo cell lysis and RNA hybridization
(https://pubmed.ncbi.nlm.nih.gov/31028633/)
- Imaging mass spectrometry (CyTOF) = measure 40 parameters of a single glass slide by using rare metal
isotopes instead of fluorochromes for labeling, allowing for highly multiplexed cellular analysis.
You measure immunophenotype in tissue to measure special connections
In the field of multiplex immunohistochemistry there is a lot of fast improvement of technologies used --> the limits of
cytometry are getting blurred!
- There are now more advanced techniques
o CODEX: imaging technique allowing multiplexed imaging of tissue by cycling through different markers
o MIBI (Multiplexed Ion Beam Imaging): Utilizes mass spectrometry to image multiple targets in a
sample.
o Imaging Mass Cytometry: Combines mass cytometry with spatial resolution to analyze tissue
architecture at the single-cell level.
o Nanostring Technologies: A method for high-throughput RNA and protein detection at the single-cell
level.
Benefits of high dimensional microscopy
1.
Multiplex tissue images can be mapped
using spatial composition analysis and the
presence of a certain interested epitope is
then checked
, 2.
Single cell segmentation can be done
by single cell analysis, where the
different cell types are found, and
the comprehensive cell states can be
reported because we can get
information on where certain
epitopes lay in the cell of interest.
3.
Neighborhood analysis can be done to
find neighboring cells of the interested
cell: here the neighborhood properties
are measured and the higher the
relation, the closer they are.
What will the future bring?
- FLIM Cytometry: integrate imaging with flow cytometry
- Single cell sequencing
o Expensive technology but if it continues to evolve and
transform and becomes affordable, this will be used
more and slowly replace flow cytometry
- Oil droplet encapsulation
o Take advantage of beads that capture mRNA in tiny lipid
droplets
o Pushing cells at a certain concentration through an oil Single cell sequencing
current causes them to be pushed into droplets
o It is possible to combine it with DNA binding
antibodies
▪ Antibodies are DNA barcoded and label
cells -> cells gel partition into droplets ->
capturing both the DNA barcode and all
RNA molecules present in cell -> added
probes specific barcode = each barcode
tells you which transcript it was and from
which cell it comes from
Oil droplet encapsulation
Elements of cytometry
- steps
o Cells in suspensions
o Push cells into a stream to get them into a single cell stream
o We direct these cells to interrogation point. Here we illuminate them with a specific beam of photons
o These excite the fluorochromes on cells
o These will change their energy state and when they relax they will emit other photons
o These photons are directed through filters into detectors → into electronics → analyzed
- Elements needed:
o Cells
o Fluidics: the system that moves cell through the instrument
o Illumination and optic to excite fluorescent markers attached to the cells and to collect
o Detector and electronics: sensors (e.g. PMTs, APDs, CCDs) that capture the emitted light and convert
into data
, o Chemistry: fluorochrome-conjugated antibodies, dyes, and proteins are used to label cells
➔ Each technology uses different elements
Conventional flow cytometry = measures physical and chemical properties of cells based on how they scatter light and
emit fluorescence. You loose a lot of material so it is not good for rare cell detection because you might lose these cells
- Chemistry
o Fluorochrome conjugated antibodies, proteins, or dyes
- Illumination by lasers
- Fluidics:
o Hydrodynamic focusing area = push cells into laminar flow, you achieve that the cells are in the middle
of the screen
o Acoustic = Put a device in middle of the stream and let it vibrate → the acoustic waves in the stream
pushes the cells in the center of the screen
o Hydrodynamic and acoustic combination is best
- Detectors and electronics
o Photon counting devices can be PMT, APD, CCD.
▪ PMT allow you to tune the sensitivity of the detectors up or down depending on your needs.
They are not very sensitive to red or infra-red, they work very well in violet and blue range.
▪ CCD are a little bit less sensitive, but they allow you to do capturing of images at the same time
Mass cytometry (CyTOF) = uses metal isotopes attached to antibodies instead of fluorochromes. These isotopes are
detected by a time-of-flight mass spectrometer.
o Chemistry
▪ Metal-conjugated antibodies = you cannot measure these with PMT, ADP, or CCD, so now you
need mass spectrometry to detect them.
o Illumination by ICP: this is an ionization source to excite them and prepare them for flow cytometry
o Fluidics
▪ To get a single cell expansion, here we use a nebulizer = gives gas to push liquid into a spray
and each droplet withing the spray has a cell
o Detector and electronics:
▪ TOF-MS: a detector in mass cytometry that measures the time it takes for ionized metal
isotopes (bound to cell makers) to travel to the detector. The distinct masses of these isotopes
allow for high-resolution, multiplexed detection of cellular features, making mass cytometry
ideal for complex immune profiling and single-cell analysis.
Single Cell sequencing = Analyzes the genetic material of individual cells to provide insights into gene expression (RNA
sequencing) or DNA variations (whole-genome or exome sequencing).
- Chemistry
o Uses DNA barcoded antibodies: The chemistry of single-cell sequencing focuses on using DNA
barcodes to capture information about both RNA (gene expression) and proteins for a more
multidimensional understanding of cellular behavior.
- Fluidics
o Microfluids: Microfluidic systems in single-cell sequencing facilitate the isolation and individual
processing of cells, ensuring that each cell's genetic material can be studied separately.
- Detection
o Sequencing: Detection in single-cell sequencing is primarily done via RNA or DNA sequencing
technologies, providing insights into gene expression (transcriptome) or genetic variations (genome or
exome).
Instruments differ in terms of spatial resolution, number of markers (multicolor possibilities), multiplexed acquisition,
fluorescence, and metal-based acquisition, autofluorescence, and specialized features such as small particle detection
and rare event analysis. Popular instruments at MCCF:
- Aurora: known for full spectral cytometry
Immunotherapy = type of treatment in which the immune system is modulated (enhanced or suppressed). There is
emphasis on cellular heterogeneity, particularly in solid tumors, where a small fraction of molecularly distinct cells such
as cancer stem cells contribute to tumor progression and therapeutic resistance.
- Single cell techniques allow you to study rare cells which is important in tumors for example to understand
their biology.
o Single cell applications
▪ Immune monitoring and immunophenotyping: identification and tracking of the immune cell
populations involved in tumor responses
▪ Cell migration and tumor killing: tracking how immune cells move and interact with tumors
▪ Structural and functional characterization: using advanced imaging techniques to understand
cellular and subcellular dynamics in real-time
- Examples of technologies:
o Cytometry and sorting which includes high-dimensional flow cytometry and live-cell sorting
o Microscopy: Advanced imaging techniques like confocal microscopy, super-resolution imaging (light-
sheet microscopy), and live-cell imaging.
o An emerging method is multidimensional data analysis which is key for handling large datasets
generated from cytometry and microscopy
The life sciences are now moving towards the trans-omics field in which we can integrate all the information of each
level
Cytometry = a technology that measures properties of single cells. It involves the quantitative analysis of physical and
chemical properties of cells or other biological particles, typically at the single-cell level.
- Parameters it measures:
o The cell surface markers are measured by fluorescence
o Light scatter measures the cell size and shape
o Using fluorescent dyes and antibodies it can also measure intracellular molecules
o And other properties of cell
- It provides correlated data that links different population profiles.
Flow cytometer = technology used to measure properties of single cells. It is a system that measures and analyzes
signals resulting from flowing particles in a liquid stream through a beam of light. It does not always deal with cells but
also chromosomes, vesicles, latex beds, or any particles that can be suspended in a fluid.
- The work comes from the Greek “cyto” (Cell) and “metry” (measurements). Early cytometers, known as FACS
(fluorescence activated cell sorters) were designed to sort cells based on fluorescence. They were the first ever
instruments made from the principle of separating cells without measuring what type of cells they really are.
- What can we do with a flow cytometer:
o Count cells
o Distinguish between biological and non-biological
o Separates live from dead
o Evaluate 105 to 107 particles in less than a minute
, o You can measure particle-scatter, autofluorescence, or fluorescence associated to other reagents
(antibodies and non-antibodies)
o Sort single particles/cells for subsequent analysis
o But also, more advanced capabilities:
▪ Obtain images
▪ Measure rare metals
▪ Measure the entire spectrum of light
Now, there are more technologies that use the principle to analyze one cell at a time but looking at different particles:
- Drop-seq dingle cell analysis
This is a platform to profile thousands of cells by encapsulating them into
individual droplets. Uniquely barcoded mRNA capture microparticles and
cells are co-confined through a microfluidic device within the droplets
where they undergo cell lysis and RNA hybridization
(https://pubmed.ncbi.nlm.nih.gov/31028633/)
- Imaging mass spectrometry (CyTOF) = measure 40 parameters of a single glass slide by using rare metal
isotopes instead of fluorochromes for labeling, allowing for highly multiplexed cellular analysis.
You measure immunophenotype in tissue to measure special connections
In the field of multiplex immunohistochemistry there is a lot of fast improvement of technologies used --> the limits of
cytometry are getting blurred!
- There are now more advanced techniques
o CODEX: imaging technique allowing multiplexed imaging of tissue by cycling through different markers
o MIBI (Multiplexed Ion Beam Imaging): Utilizes mass spectrometry to image multiple targets in a
sample.
o Imaging Mass Cytometry: Combines mass cytometry with spatial resolution to analyze tissue
architecture at the single-cell level.
o Nanostring Technologies: A method for high-throughput RNA and protein detection at the single-cell
level.
Benefits of high dimensional microscopy
1.
Multiplex tissue images can be mapped
using spatial composition analysis and the
presence of a certain interested epitope is
then checked
, 2.
Single cell segmentation can be done
by single cell analysis, where the
different cell types are found, and
the comprehensive cell states can be
reported because we can get
information on where certain
epitopes lay in the cell of interest.
3.
Neighborhood analysis can be done to
find neighboring cells of the interested
cell: here the neighborhood properties
are measured and the higher the
relation, the closer they are.
What will the future bring?
- FLIM Cytometry: integrate imaging with flow cytometry
- Single cell sequencing
o Expensive technology but if it continues to evolve and
transform and becomes affordable, this will be used
more and slowly replace flow cytometry
- Oil droplet encapsulation
o Take advantage of beads that capture mRNA in tiny lipid
droplets
o Pushing cells at a certain concentration through an oil Single cell sequencing
current causes them to be pushed into droplets
o It is possible to combine it with DNA binding
antibodies
▪ Antibodies are DNA barcoded and label
cells -> cells gel partition into droplets ->
capturing both the DNA barcode and all
RNA molecules present in cell -> added
probes specific barcode = each barcode
tells you which transcript it was and from
which cell it comes from
Oil droplet encapsulation
Elements of cytometry
- steps
o Cells in suspensions
o Push cells into a stream to get them into a single cell stream
o We direct these cells to interrogation point. Here we illuminate them with a specific beam of photons
o These excite the fluorochromes on cells
o These will change their energy state and when they relax they will emit other photons
o These photons are directed through filters into detectors → into electronics → analyzed
- Elements needed:
o Cells
o Fluidics: the system that moves cell through the instrument
o Illumination and optic to excite fluorescent markers attached to the cells and to collect
o Detector and electronics: sensors (e.g. PMTs, APDs, CCDs) that capture the emitted light and convert
into data
, o Chemistry: fluorochrome-conjugated antibodies, dyes, and proteins are used to label cells
➔ Each technology uses different elements
Conventional flow cytometry = measures physical and chemical properties of cells based on how they scatter light and
emit fluorescence. You loose a lot of material so it is not good for rare cell detection because you might lose these cells
- Chemistry
o Fluorochrome conjugated antibodies, proteins, or dyes
- Illumination by lasers
- Fluidics:
o Hydrodynamic focusing area = push cells into laminar flow, you achieve that the cells are in the middle
of the screen
o Acoustic = Put a device in middle of the stream and let it vibrate → the acoustic waves in the stream
pushes the cells in the center of the screen
o Hydrodynamic and acoustic combination is best
- Detectors and electronics
o Photon counting devices can be PMT, APD, CCD.
▪ PMT allow you to tune the sensitivity of the detectors up or down depending on your needs.
They are not very sensitive to red or infra-red, they work very well in violet and blue range.
▪ CCD are a little bit less sensitive, but they allow you to do capturing of images at the same time
Mass cytometry (CyTOF) = uses metal isotopes attached to antibodies instead of fluorochromes. These isotopes are
detected by a time-of-flight mass spectrometer.
o Chemistry
▪ Metal-conjugated antibodies = you cannot measure these with PMT, ADP, or CCD, so now you
need mass spectrometry to detect them.
o Illumination by ICP: this is an ionization source to excite them and prepare them for flow cytometry
o Fluidics
▪ To get a single cell expansion, here we use a nebulizer = gives gas to push liquid into a spray
and each droplet withing the spray has a cell
o Detector and electronics:
▪ TOF-MS: a detector in mass cytometry that measures the time it takes for ionized metal
isotopes (bound to cell makers) to travel to the detector. The distinct masses of these isotopes
allow for high-resolution, multiplexed detection of cellular features, making mass cytometry
ideal for complex immune profiling and single-cell analysis.
Single Cell sequencing = Analyzes the genetic material of individual cells to provide insights into gene expression (RNA
sequencing) or DNA variations (whole-genome or exome sequencing).
- Chemistry
o Uses DNA barcoded antibodies: The chemistry of single-cell sequencing focuses on using DNA
barcodes to capture information about both RNA (gene expression) and proteins for a more
multidimensional understanding of cellular behavior.
- Fluidics
o Microfluids: Microfluidic systems in single-cell sequencing facilitate the isolation and individual
processing of cells, ensuring that each cell's genetic material can be studied separately.
- Detection
o Sequencing: Detection in single-cell sequencing is primarily done via RNA or DNA sequencing
technologies, providing insights into gene expression (transcriptome) or genetic variations (genome or
exome).
Instruments differ in terms of spatial resolution, number of markers (multicolor possibilities), multiplexed acquisition,
fluorescence, and metal-based acquisition, autofluorescence, and specialized features such as small particle detection
and rare event analysis. Popular instruments at MCCF:
- Aurora: known for full spectral cytometry
