Light sheet fluorescence microscopy

• Light sheet microscopy – also referred to as single plane illumination

microscopy or SPIM
• Gentle way of imaging sensitive samples or fast biological processes in
vivo
• The specimen is illuminated only in a single plane at a time and
detected from the perpendicular direction.
Evolution of lightsheet Microscopy
• Sample preparation
• Building optical microscope
• imaging
SAMPLE PREPARATION
• Sample preparation step plays a crucial role in the microscopy technique to
improve the accuracy and to minimize the distortion of image acquisition.
• Type of objective is most prominent step for preparation of sample.
Ex: In light sheet microscope if, we want to analyse the volume of the
sample we will choose static method of sample preparation, if want to study
the dynamics we will opt the microfluid chip preparation.
Preparation of static sample:

Fluorescent labelled specimen

Take the capillary tube

Fill it with the supporting medium

Place the specimen in the capillary tube

Preparation of Dynamic sample:

• Microfluidic chip fabrication:

Prepare the master by using negative photosensitive material

Pore the PDMS on the master plate

Allow it solidify

Carefully remove the solidified PDMS from master plate

Puncture the holes on the PDMS for inlet and outlet

Place the resultant on the cover slip by using plasma binding

Then put the inlet and outlet tubes in the holes

Cell culture:
Take the cells of interest

Culture by using the complete medium which is suitable to it

If we want to study the mitochondria localization in the cells:
take the 80-90% confluent cell dish

Wash with PBS

Add mitotracker dye

Incubate for a hour

Then remove the dye and trypsinise the cells

Use it for study

 Light sheet based volume flow cytometry (VFC) for rapid volume reconstruction and parameter estimation on the go

Kumar, P., Joshi, P., Basumatary, J. et al. Light sheet based volume flow cytometry (VFC) for rapid volume reconstruction and parameter estimation on the go. Sci
Rep 12, 78 (2022). https://doi.org/10.1038/s41598-021-03902-8
Lightsheet characterisation
 Fluorescence based rapid optical volume screening system (OVSS) for interrogating multicellular organisms

• Multicolor flow cytometry is a useful technique when examining mixed populations of cells, such as blood and tissue
cells in human and animal samples

Basumatary, J., Ara, T., Mukherjee, A. et al. Fluorescence based rapid optical volume screening system (OVSS) for interrogating multicellular organisms. Sci Rep 11,
7616 (2021). https://doi.org/10.1038/s41598-021-86951-3
 Fluorescence based rapid optical volume screening system (OVSS) for interrogating multicellular organisms

Basumatary, J., Ara, T., Mukherjee, A. et al. Fluorescence based rapid optical volume screening system (OVSS) for interrogating multicellular organisms. Sci Rep 11,
7616 (2021). https://doi.org/10.1038/s41598-021-86951-3
 Advantage

• multichannel-array based imaging,

• cross-sectioning ability,
• organelle-level resolution,
• high throughput interrogation (due to microchannel array assembly),
• volume rendering in real-time.
Limitation
• Spatial Resolution
• Spatial resolution is a measure of the smallest object that can be
resolved by the sensor, or the ground area imaged for the
instantaneous field of view (IFOV) of the sensor, or the linear
dimension on the ground represented by each pixel.
flow cytometry
• Flow Cytometry is a technique used to detect and measure physical
and chemical characteristics of a population of cells or particles.

• capable of single-shot sectional visualization,

• high throughput interrogation,
• real-time parameter estimation,
• instant volume reconstruction with organelle-level resolution of live
specimens