MICROSCOPY Flashcards
Basic microscopy:
- Bright field illumination
- Does not revel differences in brightness between structural details (no contrast)
Basic microscopy:
* Phase-Contrast Microscopy:
Enhances contrast in transparent samples (e.g., live cells, unstained biological specimens).
Basic microscopy:
* Differential Interference Contrast (DIC) Microscopy:
Produces high-contrast, pseudo-3D images without staining.
- Fluorescence microscopy identify cells and sub-microscopic cellular components with a high degree of specificity
DIC contrast
Produces high-contrast, pseudo-3D images without staining.
- Transparent objectives can be seen using the difference in light’s refraction when transmitted through the varying thickness of the specimen
Phase contrast
Enhances contrast in transparent samples (e.g., live cells, unstained biological specimens).
Phase contrast
changes in refractive index?
- Changes in refractive index of a sample cause light rays to be shifts in phase comparted to one another
Phase contrast
minute variations
- Phase contrast translates minute variations in phase into corresponding changes in brightness in the image
Phase contrast
seeing…
morphology
Contrast by staining
- Cells/tissues can also be identifiable by specific histochemical staining and visualized by normal transmitted light
Fluorescence microscopy:
- Live-Cell Fluorescence Microscopy
(with genetically encoded fluorescent proteins): Requires no chemical fixation, allowing imaging of living cells.
- The light energy emitted is always of a longer wavelength than the light energy absorbed, due to energy lost during the transient excited lifetime
- Absorption, excitation, different state of energy, emits
* (needs to get rid of energy it just absorbed in order to return to stable)
Fluorescence microscopy:
the light spectrum
- Within the electromagnetic spectrum the “optical” spectrum regime covers the range of wavelengths from 10^-3m (infrared) to 10^-8m (UV)
- Application of this concept
- Need to know the specific wave length
- Can use multiple wavelengths at the same time
Fluorescence microscopy:
Excitation and emission
- Fluorescence is the emission of light from a compound following absorption of light of a shorter wave wavelength
- The goal is illuminating the specimen with an excitation wavelength to capture emitted light and block the reflected light
- Genetically encoded fluorescent proteins (e.g. GFP, Cherry) can be used to “tag” specific proteins in cells or fluorescent gyes can be attached to probes (antibodies or peptides) to visualize cellular structures
Fluorescent microscopy in life sciences:
- Fluorophores used in fluorescent microcopy can be divided into three classes
1) Organic dyes
2) Fluorescent protein
3) Quantum dots or nanoparticles (more niche, mostly used in drug delivery)
Fluorescent microscopy: how do you choose the right microscope technique?
wide-field - illumination
Uses broad (non-focused) light, typically from a mercury, halogen, or LED source.
Fluorescent microscopy: how do you choose the right microscope technique?
wide-field - depth of field
Captures light from all focal planes, leading to out-of-focus blur in thick samples.
Fluorescent microscopy: how do you choose the right microscope technique?
wide-field - resolution
: Limited due to light scattering from out-of-focus planes.
Fluorescent microscopy: how do you choose the right microscope technique?
wide-field - speed
Faster imaging, suitable for live-cell imaging.
Fluorescent microscopy: how do you choose the right microscope technique?
wide-field - best for
Thin samples, whole-tissue sections, and fast imaging.
Fluorescent microscopy: how do you choose the right microscope technique?
laser scanning confocal microscopy - illumination
Uses a focused laser beam that scans point by point.
* Every image is clear and in focus
* Take several images and reconstruct 3 dimensional view
* No background or foreground image
Fluorescent microscopy: how do you choose the right microscope technique?
laser scanning confocal microscopy - depth of field
A pinhole removes out-of-focus light, improving optical sectioning.
Fluorescent microscopy: how do you choose the right microscope technique?
laser scanning confocal microscopy - resolution
Higher than wide-field due to controlled depth and reduced background noise.
Fluorescent microscopy: how do you choose the right microscope technique?
laser scanning confocal microscopy - speed
Slower because it scans sequentially, but provides 3D reconstruction capabilities.
Fluorescent microscopy: how do you choose the right microscope technique?
laser scanning confocal microscopy - best for
Thick samples, high-resolution imaging, and 3D reconstruction.
Fluorescent microscopy: how do you choose the right microscope technique?
laser scanning confocal microscopy - used for
- Used for: localization/co-localization of proteins, imaging multiple fluorescent stains, 3D visualisation of stainings quantification of protein expression
- Used for: visualtion of structures between 200nm and 1cm in size which can be fluorescently labelled, cell biology, tissue biology, neurobiology
Fluorescent microscopy: how do you choose the right microscope technique?
laser scanning confocal microscopy - live animal applications
- 2 photon laser scanner confocal (LSC) microscopy
- Multiphoton (MP) microscopy in live animal applications
Fluorescent microscopy: how do you choose the right microscope technique?
wide-field vs confocal
- Wide-field microscopy is great for fast imaging but struggles with thick samples due to out-of-focus blur.
- Confocal microscopy provides clearer, high-resolution images by eliminating out-of-focus light, but is slower.
Fluorescent microscopy: how do you choose the right microscope technique?
Total Internal Reflection Fluorescence Microscopy (TIRFM)
is an advanced optical imaging technique used to visualize events occurring near the surface of a specimen with high resolution and minimal background noise.
Fluorescent microscopy: how do you choose the right microscope technique?
Total Internal Reflection Fluorescence Microscopy (TIRFM) - key principles
- Total Internal Reflection (TIR) occurs when light traveling from a medium with a higher refractive index (e.g., glass) to a lower refractive index (e.g., aqueous sample) is completely reflected at the interface beyond a critical angle.
- This creates an evanescent wave that penetrates only a few hundred nanometers (~100–200 nm) into the sample, exciting fluorophores near the surface.
- Optical phenomenon that can occur when light strokes the interface between two media of different refractive index
Fluorescent microscopy: how do you choose the right microscope technique?
Total Internal Reflection Fluorescence Microscopy (TIRFM) - adv
High Signal-to-Noise Ratio – Excites only a thin region, reducing background fluorescence.
Superficial Imaging – Ideal for studying cell membrane events and interactions near the surface.
Live-Cell Imaging – Minimizes phototoxicity and bleaching compared to full-volume illumination.
Fluorescent microscopy: how do you choose the right microscope technique?
Total Internal Reflection Fluorescence Microscopy (TIRFM) - disadv.
Limited Penetration Depth – Cannot image deeper structures beyond ~200 nm.
Requires Specialized Optics – Needs high numerical aperture (NA) objectives and precise laser alignment.
Fluorescent microscopy: how do you choose the right microscope technique?
Total Internal Reflection Fluorescence Microscopy (TIRFM) - applications
- Cell membrane dynamics (e.g., receptor-ligand interactions, endocytosis).
- Single-molecule imaging (e.g., protein tracking, molecular interactions).
- Super-resolution microscopy (e.g., STORM, PALM enhancements).
- Endocytosis-exocyotsis
- Dynamics of membrane associated proteins
- Protein arrangement
- Focal adhesions
- Growth cone migration
- Receptor-ligand interactions
- Single molecule behaviour
Fluorescent microscopy: how do you choose the right microscope technique?
light sheet microscopy (LSM)
also known as Selective Plane Illumination Microscopy (SPIM), is an advanced imaging technique that allows high-resolution, 3D imaging of biological samples with minimal photodamage.
Fluorescent microscopy: how do you choose the right microscope technique?
light sheet microscopy (LSM) - key principles
- A thin sheet of light (instead of a focused laser or full-field illumination) is used to illuminate a single optical section of the sample at a time.
- A perpendicular detection objective captures fluorescence only from the illuminated plane, reducing background noise and out-of-focus blur.
Fluorescent microscopy: how do you choose the right microscope technique?
light sheet microscopy (LSM) - adv
Low Phototoxicity & Bleaching –because spread over, Only the imaged plane is illuminated, protecting live specimens.
Fast Imaging – Parallel illumination enables rapid data acquisition.
High Resolution in 3D – Can generate detailed reconstructions of large specimens.
Fluorescent microscopy: how do you choose the right microscope technique?
light sheet microscopy (LSM) - limitations
Complex Setup – Requires specialized optics and sample mounting.(e.g. fixed to agarose)
Limited Sample Thickness – Large or opaque samples may scatter light, reducing image quality.
Fluorescent microscopy: how do you choose the right microscope technique?
light sheet microscopy (LSM) - applications
- Live embryo imaging (e.g., zebrafish, Drosophila).
- Whole-organ imaging (e.g., cleared tissues, brain slices).
- Developmental biology and cell dynamics.
- Live imagine of large samples
- Live imagine of cells dynamics (special app.)
- Imaging of large samples (w/ clearing)
- Developmental biology
Fluorescence Microscopy
description
Fluorescence Microscopy is a technique that uses fluorescent dyes or proteins to label and visualize specific structures in a sample. When excited by a specific wavelength of light, the fluorophores emit light at a longer wavelength, enabling highly specific imaging.
Fluorescence Microscopy
applications
- Cell Biology: Labeling organelles (e.g., mitochondria, nuclei, cytoskeleton).
- Live-Cell Imaging: Tracking dynamic processes like cell division and protein interactions.
- Molecular Biology: Detecting specific proteins or nucleic acids via fluorescent tagging (e.g., GFP, FISH).
- Medical Diagnostics: Identifying pathogens, cancer cells, or molecular markers using fluorescence-labeled antibodies.
Fluorescence Microscopy
advanced fluorescence techniques:
- Confocal Microscopy: Increases resolution by eliminating out-of-focus light, useful for 3D imaging.
- Super-Resolution Microscopy: Breaks the diffraction limit (e.g., STED, SIM, STORM/PALM).
super resolution microscopy
refers to advanced optical techniques that surpass the diffraction limit of conventional light microscopy (~200 nm for visible light), enabling imaging at the nanoscale resolution (10–50 nm).
Superresolution microscopy is a group of microscopy techniques that allow very small objects to be distinguished as separate identities (overcoming the diffraction barrier)
super resolution microscopy
Structured Illumination Microscopy (SIM)
is a super-resolution technique that enhances the resolution of fluorescence microscopy beyond the diffraction limit by using patterned light illumination.
super resolution microscopy
Structured Illumination Microscopy (SIM) - key principles
- A high-frequency illumination pattern (grids or stripes) is projected onto the sample.
- The interaction between the sample’s features and the pattern creates moiré fringes, which contain high-resolution information.
- By capturing multiple images with different pattern orientations and phases, a computational algorithm reconstructs a super-resolved image with up to 2× higher resolution than conventional microscopy (~100 nm resolution).
super resolution microscopy
Structured Illumination Microscopy (SIM) - adv
2× Resolution Improvement – Achieves ~100 nm lateral resolution.
Compatible with Live-Cell Imaging – Less phototoxic than STED or single-molecule techniques.
Works with Standard Fluorophores – No need for special dyes or probes.
super resolution microscopy
Structured Illumination Microscopy (SIM) - limitations
Moderate Resolution Gain – Not as high as STED or SMLM (~10–20 nm).
Computationally Intensive – Requires post-processing for reconstruction.
Sensitive to Sample Movement – Motion artifacts can distort images.
super resolution microscopy
Structured Illumination Microscopy (SIM) - applications
- Cellular structures (e.g., cytoskeleton, organelles).
- Live-cell dynamics (e.g., mitosis, vesicle trafficking).
- Neuroscience & microbiology (e.g., synaptic proteins, bacterial structures).
super resolution microscopy
STED microscopy
is a super-resolution technique that surpasses the diffraction limit by selectively depleting fluorescence outside a small focal region, achieving nanoscale resolution (~30 nm).
Creates super-resolution images by the selective deactivation of fluorophores minimising the area of illumination at the focal point and thus enhancing the achievable resolution for a given system
super resolution microscopy
STED microscopy - key principles
- Uses two lasers:
1. Excitation laser – Activates fluorophores as in conventional fluorescence microscopy.
2. STED depletion laser – A donut-shaped laser that depletes fluorescence in surrounding areas via stimulated emission, leaving only a tiny central region fluorescing. - The remaining fluorescence comes from a sub-diffraction-sized focal point, allowing super-resolution imaging.
super resolution microscopy
STED microscopy - adv
High Resolution (~30 nm) – Much better than conventional fluorescence microscopy (~200 nm).
Fast Imaging – Suitable for live-cell imaging.
Compatible with Standard Fluorophores – Works with common fluorescent dyes and proteins.
super resolution microscopy
STED microscopy - limitations
High Laser Power Requirement – Can cause photobleaching and phototoxicity.
Specialized Equipment – Requires precise laser alignment and control.
Limited Depth Penetration – Less effective for thick samples compared to two-photon or light-sheet microscopy.
super resolution microscopy
STED microscopy - applications
- Cellular structures (e.g., actin filaments, microtubules, organelles).
- Neuroscience (e.g., synaptic proteins, dendritic spines).
- Molecular interactions at the nanoscale.
super resolution microscopy
dSTORM (Direct Stochastic Optical Reconstruction Microscopy)
a single-molecule localization super-resolution technique that achieves nanoscale resolution (~10–20 nm) by stochastically switching fluorophores on and off and reconstructing a high-resolution image from their precise positions.
super resolution microscopy
dSTORM - key principles
illuminations
- Illumination of a sparse subset of fluorophores in each frame allows localisation of each individual molecule with high precision
- In this way images are built up by many frames with a small number of precise fluorophore localisations in each frame
super resolution microscopy
dSTORM - key principles
fluorescent antibodies
- Uses fluorescent antibodies to label proteins of interest (foxed samples) and it works by stochastically switching fluorophores ON/OFF causing them to “blink” so tha the localization of the blink can be detected at nm precision
super resolution microscopy
dSTORM - key principles
blinking effect
- Uses standard fluorescent dyes in a specialized buffer that induces a blinking effect, where only a small subset of fluorophores are activated at a time.
super resolution microscopy
dSTORM - key principles
how is image made
- Each fluorophore’s emission is localized with nanometer precision, and thousands of frames are recorded.
- A computational algorithm reconstructs a super-resolved image by combining all detected localizations.
super resolution microscopy
dSTORM - adv.
Ultra-High Resolution (~10–20 nm) – Better than SIM (~100 nm) and STED (~30 nm).
Uses Standard Fluorophores – No need for special photoactivatable proteins.
Single-Molecule Precision – Ideal for studying protein clusters and molecular interactions.
super resolution microscopy
dSTORM - limitations
Long Image Acquisition Time – Requires thousands of frames for reconstruction.
Photobleaching Sensitivity – Prolonged blinking cycles can limit imaging duration.
Requires Special Buffers – Oxygen scavengers and reducing agents are needed to induce blinking.
super resolution microscopy
dSTORM - applications
- Molecular organization (e.g., nuclear pores, cytoskeletal proteins).
- Membrane protein dynamics (e.g., receptor clustering).
- Neuroscience (e.g., synaptic protein mapping)
super resolution microscopy
2 colour dSTORM -
- Proteins which co-localised in conventional microscopy can be seen as distinct structures with no overlap
- The biggest advantage of dSTORM over EM is that sample prep is easy and multicolour 3D images can be taken
super resolution microscopy
expansion microscopy
- Based on the introduction of polymer gel into fixed cells and tissues and chemically-inducing swelling of the polymer by almost two orders of magnitude
ELECTRON MICROSCOPY
- Beams of electrons are used to produce images
- Wavelength of electron beam is much shorter than light, resulting in much higher resolution
It is an advanced imaging technique that uses a beam of electrons instead of light to achieve ultra-high resolution imaging at the nanometer to atomic scale.
ELECTRON MICROSCOPY
transmission (TEM)
o Electrons pass through an ultra-thin sample, forming a highly detailed 2D image.
o Electrons scatter when they pass through thin sections of a specimen
o Transmitted electrons (those that do not scatter) are used to produce image)
o Denser regions in specimen scatter more electons and appear darker
ELECTRON MICROSCOPY
transmission (TEM)
specimen preparatioon
- Specimens must be cut very thin
- Specimens are chemically fixed and stained with electron dense material
ELECTRON MICROSCOPY
transmission (TEM)
resolution
~0.1–2 nm (near atomic level).
ELECTRON MICROSCOPY
transmission (TEM)
best for
Internal structures of cells, viruses, nanomaterials.
ELECTRON MICROSCOPY
transmission (TEM)
imaging
- Produces high-resolution 2D images of internal structures.
- Can generate electron diffraction patterns for structural analysis.
ELECTRON MICROSCOPY
transmission (TEM)
applications
- Detailed internal structures of cells, organelles, viruses, and materials.
- High-resolution studies of macromolecular complexes, nanomaterials, and biological tissues.
ELECTRON MICROSCOPY
transmission (TEM)
limitations
- Requires thin samples (sample preparation can be time-consuming and challenging).
- Cannot image thick samples directly.
- Samples are generally fixed and dehydrated, so live-cell imaging is not possible.
ELECTRON MICROSCOPY
scanning (SEM)
o Electrons scan the surface of a sample, generating a detailed 3D-like image.
o Uses electrons reflected from the surface of a specimen to creat image
o Produces a 3D image of specimens surface features
ELECTRON MICROSCOPY
scanning (SEM)
resolution
~1–10 nm.
ELECTRON MICROSCOPY
scanning (SEM)
best for
Surface morphology, microstructures, biological tissues.
ELECTRON MICROSCOPY
scanning (SEM)
sample preparation
- Sample is usually coated with a conductive layer (e.g., gold or platinum) if it’s non-conductive to prevent charging under the electron beam.
- Sample doesn’t need to be thin like in TEM, so 3D surface morphology is preserved.
ELECTRON MICROSCOPY
scanning (SEM)
imaging
- Produces 3D-like images of the sample surface.
- Provides detailed information about surface features, textures, and morphological characteristics.
ELECTRON MICROSCOPY
scanning (SEM)
applications
- Imaging surface structures of biological specimens, materials, and microstructures.
- Widely used in material science, nanotechnology, semiconductor inspection, and biological applications (e.g., cell surface analysis).
Limitations:
ELECTRON MICROSCOPY
scanning (SEM)
limitations
- Does not provide detailed internal structural information (limited to surface imaging).
- Requires sample preparation (coating with conductive material) that can alter the sample’s structure.
ELECTRON MICROSCOPY
Correlative Light and Electron Microscopy (CLEM)
CLEM combines the high spatial resolution of electron microscopy (EM) with the dynamic, functional insights provided by light microscopy. It allows researchers to correlate fluorescent labeling data from light microscopy with ultrastructural details obtained from electron microscopy.
ELECTRON MICROSCOPY
Correlative Light and Electron Microscopy (CLEM)
key principles
- Light Microscopy: Provides broad overview and live-cell imaging with fluorescently labeled samples.
- Electron Microscopy: Offers detailed, high-resolution images of cellular ultrastructure at the nanometer scale.
- Correlation: After obtaining light microscopy data, the same sample is precisely positioned for electron microscopy imaging. Specialized techniques ensure accurate overlay of the two datasets.
ELECTRON MICROSCOPY
Correlative Light and Electron Microscopy (CLEM) - adv
Combines Functional and Structural Data: Light microscopy shows biological processes, while EM reveals the fine structural details.
High Resolution and Localization: Allows identification of molecules in specific cellular contexts and regions with nanometer precision.
Live-Cell Imaging Compatibility: Can observe dynamic processes (from light microscopy) and then correlate these with static high-resolution EM images.
ELECTRON MICROSCOPY
Correlative Light and Electron Microscopy (CLEM) - limitations
Complex Sample Preparation: Samples must be compatible with both light microscopy and EM, requiring multiple fixation and labeling steps.
Time-Consuming: The process of correlating data from both microscopy techniques can be lengthy.
Limited by Alignment Precision: Requires precise spatial alignment between the two imaging modalities.
ELECTRON MICROSCOPY
Correlative Light and Electron Microscopy (CLEM) - applications
- Cellular structures: Identifying protein localization with fluorescence and correlating it with ultrastructural features (e.g., organelles).
- Neuroscience: Mapping synaptic structures or protein interactions at the nanoscale.
- Nanotechnology: Visualizing engineered materials at the molecular and structural level.
simple imaging analysis and data mining
main questions to ask
- What is the scientific question we want to address
- What are the tools available
- What is the equipment available
- What is the reproducibility potential (and the costs)
simple imaging analysis and data mining
applications
- Immunolabelling
- Prganelle structure
- Protein localization and co-localization
- DNA/RNA (in situ hybridization)
- Cytochemical identification
- Oxidative metabolism
- Cell dynamics / trafficking
- Single molecule localization
- Ultrastructure
- Probe ratioing
simple imaging analysis and data mining
live staining with fluorescent dyes
- Imaging
- DIC/brightfield
simple imaging analysis and data mining
preparation and staining of specimens
- At least one factor can mess up every step
- Fixing can be very difficult because can change the composition
simple imaging analysis and data mining
fixation
- Process by which internal and external structures are preserved and fixed in position
- Process by which all cellular components inside and outside the cells keep their conformation
simple imaging analysis and data mining
fixation
heat fixing
- Preserves overall morphology but not internal structures
simple imaging analysis and data mining
fixation
chemical fixing
- Protects fine cellular substrate and morphology of larger more delicate organisms
simple imaging analysis and data mining
dyes
- Makes internal and external structures of cell more visible by increasing contrast with the background (in fluorescence and brightfield)
- Have two common features
- Chromophore groups – chemical groups with conjugated double bonds multiple colors for multiple structures
- Ability to bind cell compartment/wall
simple imaging analysis and data mining
PI - cell viability
How the assay works:
- PI cannot normally cross the cell merman
- If the PI penetrates the cell membrane it is assumed to be damaged
- Cells that are brightly fluorescent with the PI are damaged or dead
- Powerful because shows when cell is dying and when cell is alive
simple imaging analysis and data mining
preparation and staining of specimens
- Differential staining
- Divides microorganisms into groups based on their staining properties (brightfield only)
- E.g. gram stain (divides bacteria into two groups based on differences in cell wall structure)
- E.g. Acid fast satin
- Negative staining
- Used to visualise capsules surrounding bacteria that are colourless against a stained background
simple imaging analysis and data mining
preparation and staining of specimens
- simple staining
- A single staining agent is used
- E.g. haematoxylin and eosin
- Basic dyes are frequently used
- Dyes with positive charges
- E.g. crystal violet
simple imaging analysis and data mining
preparation and staining of specimens
- heavy metals
- Using high. Molecular weight probes is possible to highlight the ultrastructure of the cells within the sample
- E.g. electron microscopy
simple imaging analysis and data mining
CLEM and super resolution
- Had to optimize fixation to preserve fluorescence
- 30% of fluorescence lost
- Embedding resin carefully chosen
- Non-optimal adherence to sample
simple imaging analysis and data mining
whole sample imaging
- Embedding the sample in low melt agarose or polymers can allow visualisation of the entire morphology
- Simplest way is using agarose
simple imaging analysis and data mining
live application
Ionic flux determinations
how the assay works
- Ca2+ indicators such as OGB1 dye respond to the binding of Ca2+ by changing their fluorescence properties (in a ratiometric manner)
- The emission wavelength decreases as the probe binds available calcium
- The live cells are reacting to environmental changes
simple imaging analysis and data mining
imaging analysis
Biological stuctures present a continuous spectrum of change -> morphometry eliminates subjectivity it is more reproducible and has greater limits of detection
important definitions
- morphometry
- “the quantitative description of a structure”
important definitions
- stereology
- The extraction/interpretation of 3D data from 2D data (i.e sections of objects)
important definitions
- image processing
- Computer enhancement of a digitised image
- i.e. using various filters to remove noise, improve contrast, pseudocolouring, enhancement od regular structures (virus, crystals)
important definitions
- image analysis
- information extracted from an image
- area, perimeter, length etc.
basic image analysis
image acquisition
Capture images with appropriate resolution and settings.
basic image analysis
preprocessing
adjust contrast, brightness, and noise reduction.
basic image analysis
segmentation
Identify and separate objects of interest (e.g., nuclei, cells).
basic image analysis
quantification
Measure fluorescence intensity, object size, shape, and distribution.
basic image analysis
visualization
Use heatmaps, 3D reconstructions, or time-lapse videos.
basic image analysis
tools
: ImageJ (Fiji), CellProfiler, Python (OpenCV, scikit-image), MATLAB
data mining from microscopy data
feature extraction
Identify key parameters (e.g., fluorescence intensity, texture, morphology).
data mining from microscopy data
pattern recognition
Use clustering or machine learning to classify images.
data mining from microscopy data
big data analysis
Process large datasets using AI for automated feature detection.
data mining from microscopy data
statistical analysis
Apply statistical tests (e.g., t-tests, ANOVA) to compare conditions.
data mining from microscopy data
tools
Python (Pandas, NumPy, SciPy, TensorFlow for deep learning), R, KNIME
