Lower Cost Pump-Probe Fluorescence Microscope with High Spatial Resolution
- 詳細技術說明
- This new pump-probe fluorescence microscope provides efficient, high-resolution, lifetime imaging of ultrafast fluorescence phenomena. It provides an economical alternative to existing confocal and fluorescence microscopes.
- *Abstract
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This new pump-probe fluorescence microscope provides efficient, high-resolution, lifetime imaging of ultrafast fluorescence phenomena. It provides an economical alternative to existing confocal and fluorescence microscopes. This microscope provides superior resolution to standard fluorescence and two-photon microscopes and comparable resolution to confocal microscopes. In addition, it eliminates the problem of precision alignment that must be maintained with confocal microscopes.
DESCRIPTION/DETAILS
This pump-probe stimulated fluorescence microscope overcomes problems with existing, conventional fluorescence microscopes to provide superior spatial resolution and effective off-focal background rejection. When compared with two-photon excitation microscopes, it yields superior spatial resolution, and it offers comparable resolution with confocal microscopes. Elimination of the need for a fast optical detector for high-frequency time-resolved images provides enhanced ability to study fast molecular processes inside cellular structures, while special, sinusoidally modulated diode lasers provide a more compact, cost-effective system.
How It Works
Fluorescence lifetime imaging allows researchers to see specimen changes in response to various stimuli. While the intensity of an induced fluorescent signal has long been the primary parameter for characterizing sample structures, dynamic changes in samples over time also provide valuable information. Dynamic processes can be examined by monitoring the fluorescence induced in response to sinusoidally modulated excitation lasers. Imaging of cells throughout the lifetime of a typical chromophore is necessary to obtain useful information concerning phase and modulation; however, that lifetime is on the order of one nanosecond.
This pump-probe stimulated emission microscope obtains these phase and modulation data via two modulated lasers operating at two different frequencies. The two lasers project beams upon an autofluorescent or stained sample in a spatially overlapped fashion. The first laser (pump laser) is focused onto a diffraction-limited spot to excite a fluorescent sample, while the second laser (probe laser) is focused on the same spot to induce a stimulated fluorescent emission. Optics (e.g., scanning mirrors) combine and focus the beams to perform a raster scan, in accordance with standard video-formatting techniques.
A cross-correlation signal, generated by sample fluorescence, produces the beneficial axial sectioning and is strongly dependent upon efficient spatial overlapping of the pump and probe beams at the focal point. Spatial resolution is obtained by detecting the fluorescence at the beam frequency or its harmonics. The detector that senses the responsive fluorescence need only have a temporal resolution to sense the low-frequency, cross-correlation signal.
Output of the sensor is digitized and sampled, and its Fourier transform is taken. Image data are obtained from either the phase or modulation (or both) of the resulting signal (i.e., the signal that has been sampled and transformed). By varying the polarization of the pump beam relative to the probe beam, this design also provides a way to measure polarization relaxation of excited state molecules. Design variations also allow for three-dimensional sectioning of thick samples.
Why It Is Better
Existing fluorescence, confocal, and two-photon microscopes that typically are used in this type of research have limitations, and this pump-probe stimulated emission microscope overcomes these limitations. Standard fluorescence microscopes offer poor spatial resolution because of off-focal fluorescence interference. They also cannot provide three-dimensional section imaging. Confocal microscopes use a spatial filter in front of the optical sensor to prevent off-focal fluorescence from reaching the optical sensor. They offer much better spatial resolution than standard fluorescence microscopes and also allow for three-dimensional section imaging. The drawback is that confocal microscopes are expensive and require very precise mechanical alignment that can be difficult to maintain. Two-photon microscopes prevent off-focal fluorescence, provide three-dimensional section imaging, and alleviate the problems of alignment; however, they are very expensive.
Another problem with existing fluorescence microscopy is limitation of the temporal resolution of the optical detectors to the maximum frequency of the fluorescence phenomena from which information concerning individual fluorescence lifetimes is gathered. These microscopes fail to take advantage of the speed of the excitation laser systems that operate at frequencies up to 220 GHz.
This new microscope design eliminates the off-focal fluorescence problem and provides superior spatial resolution by detecting the fluorescence at the beat frequency or its harmonics. Compared with confocal microscopes that are expensive and require very precise mechanical alignment, this new microscope design is compact, costs less, and does not require precision maintenance of the alignment.
Furthermore, to overcome the speed limitations of conventional photomultipliers, this design uses cross-correlation frequencies that range up to the 10-kHz limit of the photomultiplier, a property which prevents the imposition of limits on the temporal resolution of the overall system and allows for imaging of ultrafast fluorescence phenomena.
Finally, this microscope's design eliminates the need for a fast optical detector for high-frequency, time-resolved images and therefore enhances the ability to study fast molecular processes inside cellular structures.
APPLICATIONS
- Fluorescence flow cytometry (e.g., DNA and chromosome analysis)
- Fluorescence microscopy in biotechnology (e.g., high-resolution study of genomics and proteomics)
- Fluorescence microscopy in medicine and healthcare (e.g., high-resolution study of DNA sequencing and chromosomes associated with disease)
BENEFITS
This pump-probe microscope costs less and provides better resolution than standard fluorescence or two-photon microscopes and comparable resolution to confocal microscopes. It allows for high-speed imaging of ultrafast fluorescence phenomena.
- Lower cost: Special sinusoidally modulated diode lasers allow for a more compact and lower cost system, providing an economic alternative to existing confocal and fluorescence microscopes.
- Superior resolution: This microscope provides resolution superior to both standard fluorescence and two-photon microscopes and comparable with confocal microscopes.
- Eliminates precision alignment: This new microscope eliminates the need for cumbersome precision maintenance of the alignment necessary with previous confocal microscope designs.
- High-speed imaging: This microscope design allows for imaging of even ultrafast fluorescence phenomena.
- Polarization relaxation measurement capabilities: This microscope provides a method for measuring polarization relaxation of excited state molecules.
For more information about this technology, please contact the University of Illinois at Urbana-Champaign Office of Technology Management at otm@illinois.edu.
- *IP Issue Date
- None
- *IP Type
- Utility
- 國家
- United States
- 申請號碼
- 5814820
- 國家/地區
- 美國
