01. Advanced Light Microscopy I

• LSFM causes far less photodamage and photobleaching

  —> LSFM illuminates only a thin plane (the light sheet) of the sample at a time, and only the region being imaged receives excitation light

  —> CFM illuminates the entire optical path above and below the focal plane, even though only one point is detected. This distributes unnecessary excitation throughout the sample.

• LSFM preserves sample integrity by minimizing light exposure to out-of-focus regions, whereas CFM exposes the entire volume, causing more damage

• non-invasive, light-based slicing of a sample to get thin, depth-resolved images

• Optical sectioning refers to the ability of a microscope to selectively image only a narrow focal plane within a thick sample while rejecting out-of-focus light from above and below that plane

• Higher NA means the lens can gather light from wider angles → higher resolution and better light-collection efficiency

• 0.1 ≤ NA ≤ 1.4–1.49

• microscope objective lenses cannot be used with just any immersion medium

• Each objective is designed for a specific refractive index (n)

  —> Objectives are optically corrected for one particular immersion medium (air, water, glycerol, or oil).

• Immersion medium affects light refraction and focusing

The orientation of the objective lens and the sample:

1. Upright microscope: Objective above the sample; light travels downward through the objective and up through the sample

   (+) deal for slides, thin sections, fixed samples, and high-resolution histology.

   (+) Objectives can get very close to the sample → excellent for high-magnification imaging.

   (+) Easier to work with thick specimens from above (e.g., tissues, whole organisms)

   (-) Not suitable for imaging cells in culture dishes or flasks, Large or thick samples may not fit between the stage and objective, Difficult to maintain sterility when imaging live cells

2. Inverted microscope: Objective below the sample; light travels upward through the objective toward the sample, which sits above it (e.g., in a dish or flask)

   (+) Designed for live-cell imaging in culture dishes, flasks, or multiwell plates; cells grow on the bottom where the objective can focus easily

   (+) Allows imaging larger or thicker samples from below, without compressing them

   (+) Better stage stability; samples remain undisturbed during long-term imaging

   (-) Generally more expensive.

   (-) Objectives have longer working distances → sometimes slightly lower maximum resolution than upright high-NA lenses

   (-) Not ideal for standard mounted slides (orientation is reversed and awkward)

   (-) More difficult to access the sample surface for manipulation from above

• A climate (environmental control) chamber in microscopy provides a stable, life-supporting environment for living samples during imaging

• main function is to keep cells, tissues, or organisms alive and physiologically stablethroughout long imaging sessions

• controlling temperature, CO₂, humidity, and sometimes O₂, allowing reliable long-term imaging and keeps the sample sterile

• GFP - a widely used genetically encoded fluorescent protein

• DAPI - a DNA-binding dye that stains nuclei (blue fluorescence)

• Alexa Fluor 488 - a bright, photostable synthetic dye used for labeling antibodies and other biomolecules

• A bandpass filter allows only a specific, narrow range of wavelengths to pass through while blocking wavelengths below and above that range

• A longpass filter allows all wavelengths longer (higher λ) than a cutoff wavelength to pass, while blocking shorterwavelengths

• Typical ranges for bandpass filters include visible light ranges like 400–600 nm

• Longpass filters can have cutoffs in the visible spectrum (e.g., >600 nm) or near-infrared

• Fluorescent dyes are used to specifically label molecules, organelles, or structures within cells and tissues so they can be visualized with high contrast under a fluorescence microscope

• absorb light at one wavelength (excitation) and emit light at a longer wavelength (emission), making the labeled structures stand out against a dark background

• Structures smaller than the resolution cannot be resolved as true shapes. They are below the diffraction limit.

• Fluorescent labeling (dyes or proteins) attaches along the length of the filament.

  —> Even though the filament is much thinner than the resolution, the fluorescence signal spreads out according to the microscope’s point-spread function (PSF).

  —> Result: the filament appears as a blurred line or “cylinder” about 200 nm wide, which is the apparent width determined by the diffraction limit, not the actual filament diameter

• Fluorescence intensity shows the presence and position of the structure, not its true diameter.

  —> Filaments appear thicker than they really are, but their overall organization, orientation, and connectivity can still be visualized.

  —> This is why researchers can study microtubule networks and actin filaments even with conventional fluorescence microscopy

• selectively excites fluorophores and directs emitted light to the detector

• controls which wavelengths excite the fluorophore and which emitted wavelengths reach the viewer

• Components:

  1. Excitation Filter

     —> Function: Selects the specific wavelength(s) of light used to excite the fluorophore.

     —> Position: In the light path before the sample.

     —> Example: For GFP, an excitation filter might pass ~480 nm light.

  2. Dichroic Mirror (Beamsplitter)

     —> Function: Reflects excitation light toward the sample while allowing emitted fluorescence (longer wavelengths) to pass through to the detector.

     —> Position: Between the objective and the light source.

     —> Acts as a wavelength-specific “traffic director.”

  3. Emission Filter (Barrier Filter)

     —> Function: Blocks residual excitation light and transmits only the fluorescence emitted by the sample.

     —> Position: Between the sample (or objective) and the detector/eyepiece.

     —> Ensures high contrast and reduces background

• Resolution depends on the ability of the lens to collect light and distinguish fine details

• Magnification simply enlarges the image; it does not improve the actual detail captured

• Spatial resolution is determined by the numerical aperture (NA), not the magnification

• Wide-field microscopy illuminates and detects everything at once, while confocal microscopy uses a scanning point and a pinhole to achieve optical sectioning and reject out-of-focus background

• Advantages of a high ratio:

  —> Higher fluorescence efficiency (better SNR)

  —> Much lower phototoxicity

  —> Much less photobleaching

  —> Better for long-term live imaging

• the pinhole only allown in focus light to pass through

• The dichroic mirror (dichroic beamsplitter)

  —> Reflects the shorter-wavelength excitation light toward the sample

  —> Transmits the longer-wavelength emission light toward the detector

• Unwanted detection of fluorescence from one channel in another channel

• Cross-talk occurs when:

  —> The emission spectrum of one fluorophore overlaps with the detection window of another fluorophore

  —> The excitation light for one fluorophore also partially excites a second fluorophore

  —> Filters (excitation/emission) or the dichroic mirror do not perfectly separate the spectra

• light intensity

• long exposure times

• photon flux

• oxygen radicals

• The NA of the illumination objective determines the trade-off between light-sheet thickness and its usable length

• A high NA creates a thin but short light sheet; a low NA produces a thicker but longer light sheet

1. Air: n≈1.00

2. Water: n≈1.33

3. Cover slip glass: n≈1.515

4. Immersion oil: n≈1.515

• A cellular spheroid is a 3D cluster of cells that self-assembles into a roughly spherical shape, often used as an in vitro tissue model

Methods

1. Hanging Drop Method

   —> Small drops of cell suspension are pipetted onto the lid of a culture dish

   —> Gravity causes cells to aggregate at the bottom of the drop

2. Low-Adhesion Plates / Ultra-Low Attachment (ULA) Surfaces

   —> Special plates coated to prevent cell adhesion

   —> Cells spontaneously aggregate into spheroids in suspension

3. Rotating / Spinner Flask Cultures

   —> Cells are cultured in suspension with gentle stirring

   —> Prevents adhesion and promotes uniform spheroid formation

Time to Form Mature Spheroids

• Typically 2–7 days to form compact, mature spheroids.

• Depends on the Cell type (some cells aggregate faster than others)

• between 500 and 5.000 cells

• the initial cell number determines:

  1. ability of the cells to form spheroids

  2. Spheroid size:

     —> More cells → larger spheroid

     —> Size affects nutrient and oxygen diffusion, mechanical properties, and experimental readouts

  3. Physiological gradients:

     —> Oxygen, nutrients, and metabolites form gradients in larger spheroids

     —> Can create hypoxic cores and necrotic regions similar to in vivo tissue

  4. Structural properties

     —> Larger spheroids have more compact cell–cell contacts and better tissue-like architecture

     —> Influences cell polarity, extracellular matrix deposition, and signaling

  5. Drug response and diffusion

     —> Drugs or dyes penetrate differently depending on spheroid size

     —> Larger spheroids may mimic resistance mechanisms seen in tumors

• Most adherent cell types can form spheroids if cultured under non-adhesive conditions, including tumor cells, stem cells, primary cells, neural cells, and co-cultures, making them versatile 3D in vitro models

• oxygen and CO2

• 3D spheroid assays provide a more physiologically relevant, tissue-like environment than 2D monolayers, improving the accuracy of studies on drug response, signaling, metabolism, and cell behavior

  —> Physiologically Relevant Cell–Cell and Cell–ECM Interactions

  —> Tissue-like Architecture

  —> More Accurate Drug Responses

  —> Improved Gene and Protein Expression Profiles

  —> Better Modeling of Dynamic Processes

  —> Enhanced Longevity

• an organoid is a miniature, 3D tissue-like structure derived from stem cells (ESCs, iPSCs, or adult stem cells) that self-organizes and differentiates to mimic the architecture and function of a specific organ

• typically derived from stem cells (adult stem cells, iPSCs, or ESCs) that are programmed to differentiate into organ-specific lineages

• Examples: intestine, brain, kidney, liver, pancreas, lung, skin, retina, and reproductive organs

• disease modeling

• drug screening/personalized medicine

• regenerative medicine/tissue engineering

• Gene editing studies

• Toxicology

• because their size, compact 3D structure, and optical properties allow fast, low-phototoxic, high-contrast volumetric imaging, making them ideal for live and multi-color studies

• most organoids are hollow and essencialy transperent so they can be easily imaged

• chain growth photocrosslinking

• Step growth photocrosslinking

• chemical process in which light energy (usually UV or visible) is used to initiate polymer formation from monomers or oligomers, resulting in a solid or gelled polymer network

• It is the fundamental principle behind SLA, DLP, and other light-based 3D-(bio)printing methods

• polymer backbone, Photoinitioator, Monomerers

• Hydrogels replace the natural ECM in physiological conditions, e.g. RGD

• To support cells, they must provide adhesion sites, nutrient permeability, appropriate mechanical properties, and optionally bioactive molecules

• Without these components, cells would not survive, proliferate, or organize properly in 3D culture

• light dose applied during photocrosslinking quantifies the energy delivered per unit area and is given by

• 3D bioprinting is crucial for regenerative medicine because it allows precise, patient-specific fabrication of 3D tissue constructs with controlled architecture, cell composition, and functionality, enabling tissue repair, organ replacement, and advanced in vitro modeling

• print pieces of functional tissues like skin or similar to treat wounds

• architecture: structure of the sample, e.g. ducts

• chemical composition and stiffness of the bioink

• Cells used in 3D bioprinting can include primary cells, stem cells, immortalized lines, or co-cultures

• Single-cell printing is possible but technically challenging

• Alternative: print cell aggregates or spheroids, which improve viability and mimic tissue architecture from the start

  —> like a reservoir of cells that then can distribute throughout the printout

• This strategy is especially common in tissue engineering and regenerative medicine, where functional tissue requires multicellular organization