ENDOSCOPIC MASS SPECTROSCOPY WITH MOVING SAMPLING STRING
The first demonstration of the present sampling concept was conducted using a crude arrangement as shown in Fig. 4a. In this preliminary test, two transparent glass tubes were used as the guiding capillaries for the cotton thread to wipe the marking made with a red marker pen on the finger locally. Household cotton thread (size No. 20, approx. dia.: 0.3 mm) typically used for hand sewing was used as received here. The rubbing effect on the finger could be felt by the experimenter when the thread was rolled continuously. The photograph taken after the sampling (Fig. 4b) indicated that a small portion of the red marking was wiped off from the surface. To evaluate if the sample had sufficiently adhered to the cotton thread, a simple liquid extraction experiment was conducted using ESI with water/methanol as solvent. In Fig. 5a, the sampling string probe was touched by the red inked finger, and the string was moved with a set of pulleys to the proximity of the ion inlet of the MS and intercepted by the spray from a pneumatically assisted ESI source. Rhodamine (Rhodamine B/Rhodamine 6G) from the red ink was detected as [M−Cl]+ as shown in Fig. 5b. In this measurement, the sampling probe was touched three times with approximately equal time interval and three peaks from rhodamine showed up in the ion chronogram of [M−Cl]+ in Fig. 5c.
|Fig. 4. a) Preliminary test of the sampling concept using a moving cotton thread. The guiding capillaries here are made of two glass tubes adhered together by the green plastic tape. The velocity of the moving thread is approximately 0.3 m/s. The sampling time is a few seconds. b) Photograph showing the effect of sampling. A part of the region marked by red ink is wiped off the surface and sampled by the moving cotton string.|
|Fig. 5. Remote sampling of dried red ink deposited on a finger. a) Photograph showing the touching of the sampling probe with the finger. The touching is performed three times. b) Mass spectrum of the rhodamine from the red ink. c) The chronogram of the selected ion from rhodamine.|
A prototype of a working mass spectrometric endoscope was constructed using a low-cost industrial endoscope (STS, Nagoya, Japan).33) Photograph of the constructed prototype is shown in Fig. 6. The flexible cable of the endoscope was 5.5 mm in diameter and a camera (720×480 pixels) and the miniature super-bright LEDs were attached at the distal end of the endoscope. The spool of cotton thread was loaded to a spool pin and the drawing of the thread was driven by a DC motor. Two flexible plastic tubes (o.d.: 4 mm, i.d.: 2 mm) were attached to the endoscope for the guiding of the cotton thread. A partially cut metallic tube (i.d.: 0.8 mm) was folded to form a V-shape guiding capillary and the cotton thread within the guiding capillary was exposed to the exterior at the probe tip. The tip of the sampling probe was positioned at the focal point of the camera lens which was approximately 10 mm from the end of the endoscope.
|Fig. 6. Prototype of a working endoscopic probe for in-situ mass spectrometry that incorporates an industrial endoscope. The ion source is not installed in this photograph (see Fig. 8). Inset shows the close-up view of the tip of the sampling probe.|
To sample materials from the surface, the probe tip was slightly pressed onto the surface, and the moving cotton thread would wipe and carry the adhered sample along with it. Effects of sampling using this prototype on soft biomaterials are shown in Fig. 7. In Fig. 7a, it was a soft leaf put on a white paper. With appropriate drawing speed for the cotton thread and a gentle pressing, craters of approximately 0.3×0.8 mm were formed on the sample surface. In Fig. 7b, the sample was a piece of chicken liver purchased from the local wet market. In this case, the pressing force was higher than that in Fig. 7a to magnify the sampling effect. The crater it created was clearly visible with bare eyes, and the staining of the sampling string was also visible.
|Fig. 7. View from the endoscope camera during the sampling on a) soft leaf, and b) chicken liver.|
The thread was drawn at a speed of approximately 0.3 m/s to move across an extraction and ionization region. The distance between the string and the ion sampling orifice was approximately 4 to 5 mm. The arrangements of the ion sources used in this system are shown in Fig. 8. Two methods: liquid extraction and thermal desorption were used for the desorption and ionization. In the liquid extraction, the electrospray emitter was position horizontally with the emitter was directed towards the ion inlet (Fig. 8a). The moving cotton string was placed in between the ESI sprayer and the ion inlet. This configuration resembled the arrangement of a transmission desorption electrospray ion source.39) The ESI sprayer was custom-made from two coaxial metallic capillaries with a 100 μm gap between them. The i.d. for the ESI emitter was 100 μm. The pneumatic gas (compressed air) was supplied from an air compressor (Anest Iwata, Yokohama, Japan) and the typical flow rate of gas was 3 L/min. The cotton thread was splashed with fast flowing charged droplets and the ions were formed by secondary electrospray from the thread. Chloroform/methanol 7 : 3 v/v doped with ammonium acetate was used as a solvent to optimize the extraction of lipid components. The solvent flow rate was 20 μL/min. In Fig. 8b, a gas heater was used to generate hot air to heat the cotton thread rapidly. The air flow rate was 2 L/min and the air temperature was 210°C. The thermally desorbed compounds were ionized by APCI using corona discharge. The corona discharge needle was from the original APCI ion source (Thermo Fisher Scientific).
|Fig. 8. Ion sources used for the extraction and ionization for the sample adhered to the sampling string. a) Transmission desorption electrospray. b) Thermal desorption atmospheric pressure chemical ionization.|
Using the MS endoscope, in-situ and in-vivo analysis on the liver of a living mouse was performed using ESI and APCI as shown in Fig. 9a. The mice used in the experiment were deeply anesthetized with 50 mg/kg pentobarbital sodium salt, followed by laparotomy to expose the liver. After the surgery, they were held supine on the sample plate for measurement. The mass spectrum acquired from the mouse liver is shown in Fig. 9b. The mass spectrometer here was a benchtop Orbitrap (Exactive, Thermo Fisher Scientific, Bremen, Germany). The sampling was performed three times at different spots and the selected ion chronogram for a particular lipid (phosphatidylcholine, PC[34 : 2]) is depicted in the inset of Fig. 9b. The probe was held by operator’s hand, therefore, owing to vibration, fluctuation in ion signal was expected. By performing the continuous sampling, the time profile of ion signals could, in principle, reflect the depth profile for the chemical contents on a particular spot. By switching the ion source, the acquired APCI mass spectrum of the same liver is depicted in Fig. 9c. As expected, the detected volatile compounds were of lower molecular weight and the most abundant species were originated from cholesterol and retinol.
|Fig. 9. In-situ and in-vivo endoscopic mass spectrometry on the liver of a living mouse. a) Photograph showing the sampling process. b) ESI mass spectrum. Inset shows the selected ion chronogram for a particular lipid. B) APCI mass spectrum. Insets show the selected ion chronogram for retinol and cholesterol. b) and c) are adapted from ref. 33.|
For thermal desorption, it was found that the alignments for sampling string, gas heater, and corona needle were not critical to achieve an optimal sensitivity. As for the liquid extraction in the ESI method, however, the position and the orientation of the sprayer relative to the string and ion inlet were critical. If the sample was not homogeneously spread over the sampling string, the reproducibility of the ion signal also depended on which side the sample adhered to the sampling string. Improvement on the ESI based ionization and extraction method is underway and will be followed by future publications.