Label-free detection of mitochondrial distribution in cells by nonresonant Raman microspectroscopy

Abstract

High spatial resolution Raman maps of fixed cells in an aqueous environment are reported. These maps were obtained by collecting individual Raman spectra via a Raman microspectrometer in a raster pattern on a 0.5-microm grid and assembling pseudocolor maps from the spectral hypercubes by multivariate methods. The Raman maps show the nucleus and the nucleoli of cells as well as subcellular organization in the cytoplasm. In particular, the distribution of mitochondria in the perinuclear region could be demonstrated by correlating distinct areas of the Raman maps with corresponding areas of fluorescence maps of the same cells after staining with mitochondria-specific labels. To the best of our knowledge, this is the first report of label-free detection of mitochondria inside a somatic mammalian cell using Raman microspectroscopy.

FIGURE 1

(A) Bright-field microscopic image of a HeLa cell in buffer solution, 20× objective. (B) Integrated Raman intensities in the 2800–3000 cm⁻¹ region of the cell shown in A, collected at a dwell time of 0.5 s/point and a point spacing of 0.5 μm. The spectra were excited by ∼10 mW at 488-nm laser radiation. Bright yellow hues indicate highest, and orange hues low integrated C-H stretching intensities. (C) Nine cluster pseudocolor Raman map, based on the Raman data shown in B. Data were vector normalized and correlated in the 1200–1800 cm⁻¹ region.

FIGURE 2

(A–C) Fluorescence images of three HeLa cells after staining with green fluorescent Mitotracker stain. Excitation wavelength: 488 nm, integrated fluorescence intensity between 510 and 540 nm. Bright green hues indicate highest fluorescence intensities. (D–F) Five cluster Raman maps of the same cells, taken before addition of Mitotracker stain and fluorescence measurement. Notice the congruence of the light green areas in the Raman and the fluorescence maps.

FIGURE 3

Raman spectra of the nuclei of cells 1, 2, and 3 shown in Fig. 2 (D–F), extracted as the mean cluster spectra of all clusters shown in gray in Fig. 2.

FIGURE 4

Mean cluster spectra of the nuclei (black), mitochondrial areas (medium gray), and cytoplasm (light gray) of the cells shown in Fig. 2. Between 600 and 1800 cm⁻¹, the intensities were expanded by a factor of 2.5 to better demonstrate the spectral differences between the three regions.