Microscopy of fluorescently labeled proteins has become a standard technique for live cell imaging. However, it is still a
challenge to systematically extract quantitative data from large sets of images in an unbiased fashion, which is
particularly important in high-throughput or time-lapse studies. Here we describe the development of a software package
aimed at automatic quantification of abundance and spatio-temporal dynamics of fluorescently tagged proteins in vivo in
the budding yeast Saccharomyces cerevisiae, one of the most important model organisms in proteomics. The image
analysis methodology is based on first identifying cell contours from bright field images, and then use this information to
measure and statistically analyse protein abundance in specific cellular domains from the corresponding fluorescence
images. The applicability of the procedure is exemplified for two nuclear localized GFP-tagged proteins, Mcm4p and
The average environmental response of red blood cells (RBCs) is routinely measured in ensemble studies, but in such investigations valuable information on the single cell level is obscured. In order to elucidate this hidden information is is important to enable the selection of single cells with certain properties while subsequent dynamics triggered by environmental stimulation are recorded in real time. It is also desirable to manipulate and control the cells under phsyiological conditions. As shown here, this can be achieved by combining optical tweezers with a confocal Raman set-up equipped with a microfluidic system. A micro-Raman set-up is combined with an optical trap with separate optical paths, lasers and objectives, which enables the acquisition of resonance Raman profils of single RBCs. The microfluidic system, giving full control over the media surrounding the cell, consists of a pattern of channels and reservoirs produced by electron beam lithography and moulded in PDMS. Fresh Hepes buffer or buffer containing sodium dithionite are transported through the channels using electro-osmotic flow, while the direct Raman response of the single optically trapped RBC is registered in another reservoir in the middle of the channel. Thus, it is possible to monitor the oxygenation cycle in a single cell and to study photo-induced chemistry. This experimental set-up has high potential for monitoring the drug response or conformational changes caused by other environmental stimuli for many types of single functional cells since "in vivo" conditions can be created.
We introduce a novel setup combining a micro-Raman spectrometer with external optical tweezers, suitable for resonance Raman studies of single functional trapped cells. The system differs from earlier setups in that two separate laser beams used for trapping and Raman excitation are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually to optimize the functionality of the setup and to enable the recording of resonance Raman profiles from a single trapped cell. Trapping is achieved by tightly focusing infrared (IR) diode laser radiation (830 nm) through an inverted oil-immersion objective, and resonance Raman scattering is excited by the lines of an argon:krypton ion laser. The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using different excitation lines (488.0, 514.5, and 568.2 nm) in resonance with the heme moiety and by studying spectral evolution during illumination. We found that great care has to be taken in order to avoid photodamage caused by the visible Raman excitation, whereas the IR trapping irradiation does not seem to harm the cells or alter the hemoglobin Raman spectra. Stronger photodamage is induced by Raman excitation using 488.0- and 514.5-nm irradiation, compared with excitation with the 568.2-nm line.
It has recently been shown that the combination of Raman spectroscopy and optical tweezers constitute a powerful tool for biological studies. Raman spectra of single cells immobilized in a sterile surrounding can then be recorded without the risk of surface-induced morphological cell changes. Further, the complete cellular environment can be changed while measuring dynamics in real time. We here introduce a novel Raman tweezers set-up ideal for resonance Raman studies of single cells. The system differs from earlier set-ups in that two separate laser beams, used for trapping and Raman excitation, are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually, for example in order to optimize the functionality of the set-up or to record resonance Raman profiles from the same trapped cell. Further, the tweezers can be removed from the system without affecting the spectrometer configuration. Trapping is achieved by tightly focusing IR diode laser radiation (830 nm) through an inverted oil immersion objective with high numerical aperture (NA = 1.25), while Raman scattering is excited by the lines of an ArKr ion-laser. The backscattered Raman signal is collected by a single-grating spectrometer equipped with a microscope and a 60x water-immersion objective (NA = 0.9). The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using differen excitation lines (488, 514.5 568.2 nm) in resonance with the heme moiety and by studying the spectral evolution during illumination.