Cell Biology JoVE (Open Access) Citable · DOI

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

Name: Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation
Uploaded: 2026-06-17
Duration: 15 min 27 s
Description: The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

DOI: 10.3791/52208-v

What you'll learn

✓ Prepare agarose pads for live-cell fluorescence microscopy imaging
✓ Internalize fluorescent biomolecules into microorganisms using electroporation
✓ Acquire and analyze fluorescence microscopy data from living cells
✓ Quantify photobleaching and fluorescence dynamics in single cells

Protocol

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

Difficulty

advanced

Total time

~3–4 hours per sample batch (including microscopy acquisition and analysis)

Model organism

Microorganisms (genus/species not specified in abstract)

Biosafety

BSL-1

Steps

Prepare agarose pad substrate for microscopy

Prepare and mount agarose pads on microscope slides to immobilize living microorganisms during imaging. This provides a stable mounting medium that maintains cell viability during fluorescence microscopy.

▶ 02:36

Internalize fluorescent biomolecules via electroporation

Apply electrical pulses to introduce fluorescent DNA or proteins into living microorganisms. Electroporation creates transient pores in the cell membrane, allowing uptake of exogenous biomolecules.

▶ 03:29

Acquire fluorescence microscopy images of living cells

Capture time-series fluorescence images using appropriate filters and settings to visualize internalized biomolecules in live microorganisms. Record multiple optical sections or frames to document spatial and temporal distribution.

▶ 07:14

Perform general fluorescence image data analysis

Process and analyze acquired microscopy data to quantify fluorescence intensity, localization, and spatial patterns. Apply image segmentation and intensity measurements to characterize biomolecule distribution.

▶ 09:07

Quantify photobleaching and fluorescence dynamics

Measure fluorescence intensity loss over time (photobleaching) in single cells to assess biomolecule stability and compartmentalization. Use region-of-interest analysis to track temporal changes in fluorescent signal.

▶ 10:42

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