Raman Microscopy:

Raman microscopy is a powerful analytical technique that combines Raman spectroscopy with
optical microscopy to provide detailed chemical and structural information about a sample at a
microscopic level.

Principle of Raman Microscopy

The principle of a Raman microscope is based on Raman spectroscopy which involves the
inelastic scattering of light (Raman scattering). A laser beam is directed onto the sample. The
light interacts with the molecular vibrations of the [Link] of the light is elastically
scattered (Rayleigh scattering), but a small fraction is inelastically scattered, resulting in a shift
in energy (Raman scattering). The inelastically scattered light is collected and analyzed to
produce a Raman spectrum, which acts as a "chemical fingerprint" of the [Link] technique
is widely used for chemical analysis, material characterization, and even biological studies.

Sample Preparation

Sample preparation for Raman microscopy is relatively simple but dependson the type of sample
and the desired analysis

1. Solid Samples: Ensure the surface is clean and flat to avoid scattering artifacts. Powders
can be pressed into pellets or placed on a glass slide. Avoid fluorescent materials that
may interfere with Raman signals.
2. Liquid Samples: Place the liquid in a cuvette or on a glass slide with a coverslip. Ensure
the sample is transparent to the laser wavelength.
3. Biological Samples: Fixation or freezing may be required to preserve the sample.
4. Special Considerations: Minimize fluorescence by choosing an appropriate laser
wavelength. Avoid overheating or damaging the sample by optimizing laser power.

Operation:

Operating a Raman microscope involves several steps to ensure accurate and reliable results:

1. Laser Selection: Choose a laser wavelength that minimizes fluorescence and maximizes
Raman signal.
2. Calibration: Calibrate the instrument using a standard
3. Microscope Objective: Select an appropriate objective lens to focus the laser on the
sample.
4. Focus the Laser: Use the microscope to focus the laser on the area of interest.
5. Adjust Laser Power: Optimize laser power to avoid sample damage while maintaining a
strong Raman signal
 6. Collect Spectra: Acquire Raman spectra in point mode, line scan, or mapping mode for
spatial distribution analysis.
7. Baseline Correction: Remove background fluorescence or noise.
8. Peak Identification: Compare Raman peaks with spectral libraries to identify chemical
components.
9. Mapping and Imaging: Generate chemical maps to visualize thedistribution of
components in the sample.

Two-Photon Microscopy

Two-photon microscopy (TPM), also known as two-photon excitation microscopy, has

revolutionized fluorescence imaging by enabling high-resolution visualization of living tissues at
unprecedented depths. This technique overcomes key limitations of traditional fluorescence and
confocal microscopy, particularly in reducing photo-toxicity and improving penetration in thick
biological samples.

Principle of Two-Photon Microscopy

Two-photon microscopy operates on the principle of two-photon excitation, where a fluorophore

absorbs two lower-energy (longer-wavelength, typically near-infrared) photons simultaneously
to reach an excited state, after which it emits a single higher-energy photon. This process
requires the two photons to arrive at the fluorophore within an extremely short time frame
(around 1 femtosecond) and occurs only at the focal point of a high-intensity laser beam due to
its nonlinear nature. The confined excitation minimizes out-of-focus fluorescence and
photodamage, enabling high-resolution, deep-tissue imaging with reduced phototoxicity, making
it ideal for studying live biological samples in three dimensions.

Sample preparation

Sample preparation for two-photon microscopy is critical to ensure high-quality imaging while
maintaining the viability of live tissues or cells. Following steps are followed while preparing the
sample for it:

1. Sample Selection & Preparation: TPM is used for both live and fixed samples. Live-cell
imaging requires special care to maintain physiological conditions. Thick tissue sections (100–
500 µm) are used instead of thin sections since TPM enables deeper penetration.

2. Staining & Labeling: Common fluorophores include GFP, YFP, and synthetic dyes like
Alexa Fluor or Rhodamine are employed for the labelling of the samples. If targeting specific
proteins, antibodies conjugated with fluorescent dyes are used. Some tissues (like collagen or
NADH) exhibit autofluorescence, eliminating the need for dyes.

3. Mounting the Sample: Cells are typically maintained in special chambers with physiological
media (e.g., DMEM, RPMI) and controlled temperature (37°C). Tissues are embedded in
agarose or gel and placed on coverslips for stability.
4. Immersion Medium: Water-Immersion is often used for live samples to match refractive
index and reduce aberrations. Oil or Glycerol immersion is used for fixed samples when deeper
imaging is required.

5. Imaging Considerations: Adjust laser power to minimize photobleaching. Increase power for
deeper tissue penetration if needed. Apply deconvolution or image processing for enhanced
clarity.

Operation
Two-photon microscopy is operated using a specialized setup that includes an ultrafast pulsed
laser, scanning optics, detectors, and advanced software for image acquisition and analysis.

1. Laser Excitation: An ultrafast pulsed laser (usually a titanium-sapphire laser) emits

near-infrared (NIR) light in femtosecond pulses. This laser provides the high peak
intensity required for two-photon excitation.
2. Beam Scanning: The laser beam is directed through a set of scanning mirrors
(galvanometers) that raster-scan the beam across the sample in a precise pattern, point by
point.
3. Objective Lens: The scanned laser beam is focused onto the sample through a high
numerical aperture (NA) objective lens, which concentrates the light to a tiny focal spot.
The high intensity at the focal point enables two-photon excitation.
4. Fluorescence Emission: At the focal point, fluorophores in the sample simultaneously
absorb two photons and emit a single higher-energy photon (fluorescence). This emission
occurs only at the focal plane due to the nonlinear nature of two-photon absorption.
5. Detection: Emitted fluorescence is collected by the same objective lens and directed
through a dichroic mirror or filter to separate it from the excitation light. The
fluorescence is then detected by a sensitive photomultiplier tube (PMT) or other
detectors.
6. Image Formation: The detected fluorescence signals are synchronized with the scanning
mirrors to build a 2D image pixel by pixel. By adjusting the focal plane, 3D images can
be reconstructed by stacking multiple 2D slices.
7. Software Control: A computer controls the laser, scanning mirrors, and detectors,
allowing for precise adjustment of imaging parameters (e.g., laser power, scan speed,
depth). Specialized software processes the data to generate high-resolution images and
videos.

Holographic Microscopy:
Holographic microscopes are revolutionary tools that use digital holography to record and
reconstruct images of microscopic objects. Unlike traditional microscopes, holographic
microscopes capture the light wave front information from the object, allowing for quantitative
phase imaging (QPI) and three-dimensional reconstruction of the object.
Principle
Holographic microscopy is an advanced imaging technique that records the interference pattern
(hologram) of light scattered from a sample. It captures both the amplitude (intensity) and phase
information of light, unlike traditional microscopy which only captures intensity. This allows for 3D
imaging and quantitative analysis of transparent or semi-transparent biological and non-biological
samples.
Interference: When a laser beam is split into two parts—one interacts with the sample (object beam) and
the other is used as a reference (reference beam)—they recombine to form an interference pattern.
Hologram: This interference pattern encodes information about the sample’s shape, structure, and
refractive index.
Reconstruction: The hologram is recorded on a digital sensor and processed by a computer to recreate a
3D image of the sample.

Sample Preparation
A. Biological Samples
Live Cells: No staining is needed. Use culture media compatible with imaging.
Fixed Cells: Mount samples on slides in a refractive index-matching medium.
Tissue Sections: Thinly sectioned samples (~5-20 µm) are prepared for clear phase imaging.

B. Non-Biological Samples:
Nanoparticles, colloids, and microstructures can be dispersed in a transparent medium (e.g., water or oil).

Operation:
Step 1: Illumination:
A coherent light source (usually a laser) generates a beam.
The beam is split into two:
Object Beam: Passes through or reflects from the sample.
Reference Beam: Travels directly to the detector without interacting with the sample.
Step 2: Interference and Recording:
The object and reference beams are combined, forming an interference pattern (hologram) on a digital
camera (e.g., CCD or CMOS sensor).
This pattern contains both phase and intensity information of the sample.
Step 3: Image Reconstruction:
A computer applies mathematical algorithms (e.g., Fourier Transform) to reconstruct the 3D image.
The reconstructed image can be digitally focused at different depths without physically adjusting the
microscope.