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Updated: Feb 6, 2026
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Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer
Published on: November 12, 2021
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A Fluorescence Correlation Spectrometer for Measurements in Cuvettes.
Bankanidhi Sahoo1, Timir Baran Sil1, Biswajit Karmakar2
1Tata Institute of Fundamental Research, Serilingampally, Hyderabad, India.
Biophysical Journal
|August 9, 2018
Summary
We developed a new fluorescence correlation spectroscopy (FCS) setup for cuvettes, enabling single-molecule measurements. This versatile cuvette FCS offers enhanced temperature range and accuracy for protein unfolding studies.
Area of Science:
- Biophysics
- Spectroscopy
- Single-molecule analysis
Background:
- Microscope-based fluorescence correlation spectroscopy (FCS) has limitations in temperature range and experimental flexibility.
- Conventional FCS setups often require specialized objectives and sample preparation.
Purpose of the Study:
- To develop a novel fluorescence correlation spectroscopy (FCS) setup adaptable for standard cuvettes.
- To enable single-molecule measurements with enhanced capabilities beyond traditional microscope-based FCS.
Main Methods:
- Utilized a horizontally mounted, 0.7 NA, extra-long working distance air objective (>1.8 mm working distance).
- Designed a cuvette-based FCS setup, optimizing for cuvette quality and alignment.
- Achieved radial resolution of ~340 nm and an observation volume of 1.8 fL.
Main Results:
- Demonstrated high sensitivity to cuvette quality and alignment.
- Achieved molecular brightness >44 kHz and signal/noise ratio >110 for rhodamine B.
- Validated performance with radial resolution of ~340 nm and effective observation volume of 1.8 fL.
Conclusions:
- Cuvette FCS offers a wider temperature range compared to microscope-based FCS.
- Enables accurate studies of protein unfolding via coupling with automatic titrators.
- Provides ease of use and accessory compatibility for spectrofluorometer-like applications.
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