Simultaneous Topographic and Fluorescence Imagings of Recombinant Bacterial Cells Containing a Green Fluorescent Protein Gene Detected by a Scanning Near-Field Optical/Atomic Force Microscope

Abstract

A scanning near-field optical/atomic force microscope (SNOAM) system was applied for simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic force microscopy cantilever and as a scanning near-field optical microscopy probe. Optical resolution of this system was about 50-100 nm in fluorescence mode for fluorescent latex beads on a quartz glass plate. Green fluorescent protein (GFP) is a convenient indicator of transformation and should allow cells to be separated by fluorescence-activated cell sorting. The gene coding to GFP was cloned in recombinant Escherichia coli. The SNOAM system used 458- or 488-nm irradiation from a multiline Ar ion laser for excitation of GFP, since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluorescence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. A nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, it can be seen that individual E. coli cells expressed different fluorescence intensities. Fluorescence obtained by SNOAM indicated that GFP oxidation possibly occurred near the cell surface. A SNOAM system also indicated the possibility of precise imaging of native cells in liquid.

Flow cytometric measurement of fluorescence (Förster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single‐laser excitation at 458 nm

“…The argon-ion laser tuned to a wavelength of 458 nm recently was reported to have appropriate energy to excite CFP, YFP, and GFP simultaneously with sufficient separation of their respective emission signals (37)(38)(39)(40). However, YFP is not optimally stimulated by the 458-nm line of argon-ion laser in comparison with its excitation with the 488-nm line of the argon-ion laser.…”

“…When an excitation wavelength of 458 nm was used, the optical settings were modified from a previously described method (37)(38)(39)(40), as shown in Figure 1. Briefly, the enhanced CFP fluorescence signal in FL2 was separated from YFP fluorescence with a 530-nm shortpass (SP) dichroic filter, reflected by a 500-nm longpass dichroic filter, and collected with a 480/30-nm bandpass (BP) filter.…”

Probe microscopy methods and applications in imaging of biological materials

“…In addition, other imaging modalities can be combined with AFM to provide more comprehensive information about cellular processes: Gold-and silver-coated AFM tips are known to strongly enhance Raman signals, and nucleic acids [24], proteins [25], bacterial [26,27] and eukaryotic cell surfaces [28], and sectioned erythrocytes [29] have been investigated using a combination of AFM and Raman spectroscopy using TERS-compatible tips, even allowing the nucleotide-level detection in DNA strands [30]. Similarly, AFM can be performed alongside other scanning probe techniques such as scanning near-field optical microscopy (SNOM) [31], and AFM tip-based nanoneedles and nanoscalpels have also been fabricated for performing highly precise measurements in living cells [32][33][34].…”

High frequency-bandwidth optical technique to measure thermal elongation time responses of near-field scanning optical microscopy probes

“…NSOM has been applied in diverse fields 3 ranging from high-density data storage, 4,5 light pulse propagation in photonic structures, 6 and charge carrier dynamic studies in semiconductors, 7,8 to imaging biological samples. 9,10 It has also proved single molecule sensitivity at room temperature. 3,11 Presently, there is much interest in improving NSOM probes throughput efficiencies to enable widespread use of NSOM in areas that require high intensity light sources.…”