2.1.

Mirrors

Mirrors consisted of a round standard cover glass (thickness $0.19\text{to}0.22\mathrm{mm}$ , diameter $11.8\mathrm{mm}$ ; E. Hefele Medizintechnik, Munich, Germany), coated with aluminum vapor (performed by NiWe Decor GmbH, Kaufbeuren, Germany). This technique of mass coating is used, e.g., in manufacturing costume jewelry and is thus a low-cost procedure, in the range of 20 Euro-cents or less per mirror.

2.2.

Microscope Setup

Microscopic observations were performed on a TriMScope (LaVision Biotec, Bielefeld, Germany) built around an Olympus BX 51 microscope (Olympus, Hamburg, Germany) and equipped with a tunable Ultra II Titan:Sapphire laser (Coherent, Dieburg, Germany) and an optical parametric oscillator (OPO; APE, Berlin, Germany) that is pumped by the Ti:Sa. The beam multiplexer of the TriMScope was not used in the current study. While the Ti:Sa excitation beam consists of two parts that are polarized orthogonal to each other, the OPO-generated beam is polarized in one direction only.

The OPO was used to generate $1270\mathrm{nm}$ light $\left(700\mathrm{mW}\right)$ . The unattenuated intensity at the sample (i.e., after the objective) was measured with $250\text{to}300\mathrm{mW}$ . An Olympus XLUMPlanFl $20\times ∕0.95\text{-}\mathrm{W}$ objective was used (working distance $2\mathrm{mm}$ ). The following detection channels were used for backward (epi) detection: blue $\left(417\text{to}477\mathrm{nm}\right)$ , orange $\left(550\text{to}600\mathrm{nm}\right)$ , and red $\left(604\text{to}644\mathrm{nm}\right)$ . An additional blue channel was available in the forward direction. $700\text{-}\mathrm{nm}$ short-pass filters blocked out excitation light. Light collection in the forward direction was performed by an Olympus WI-UCD condenser, NA 0.8. If not required for forward detection, the condenser was removed to avoid reflection of light at its surfaces. Photomultiplier tubes (PMTs) were Hamamatsu H6780-01 for the blue channels, H6780-20 for the others.

2.3.

Fluorescent Test Slides

Fluorescent plastic slides from Chroma Technology (Rockingham, Vermont) were used. These slides have a high batch-to-batch variability of their excitation and emission behavior. Also, the excitation spectrum is not continuous but shows alternating maxima and minima. Thus, a suitable excitation wavelength must be determined for each slide. We used the following settings: blue slide, $800\text{-}\mathrm{nm}$ excitation, detection in blue channel; red slide, $1040\mathrm{nm}$ , detection in orange channel. Mirrors and uncoated control coverslips were attached to the underside of the slide with adhesive tape. 3-D stacks were recorded starting above and stopping below the slide. Actual thickness was determined with a sliding caliper to be $1.8\mathrm{mm}$ . The optical thickness—i.e., the distance the stage had to be moved until the focus plane reached the bottom of the slide—was about $1.45\mathrm{mm}$ , due to refraction index mismatch between the immersion water and the plastic slide.¹⁸ To obtain the true difference from the surface of the slide to the focal plane, depths have thus to be multiplied by $1.8∕1.45=1.24$ . Images were recorded with $500\mu \mathrm{m}$ , $512\text{pixels}$ (px), $800\mathrm{Hz}$ , with 20- or $50\text{-}\mu \mathrm{m}$ $z$ distance. The measured average intensity of each section was calculated with ImageJ 1.41o, a public-domain software (W.S. Rasband, U.S. National Institutes of Health, Bethesda, Maryland; http://rsbweb.nih.gov/ij/).

2.4.

Collagen Gel

Collagen gel (PureCol, Advanced BioMatrix, San Diego, California ) preparation was done according to Reichardt ¹⁹ with minor modifications. A semicircle-shaped mirror and a respective uncoated control coverslip were placed side by side on a microscopic slide, and the gel was cast on top, using a rubber o-ring as barrier. The optical thickness (see earlier) of the gel varied and was $1300\text{to}1450\mu \mathrm{m}$ . SHG recording was performed in the blue channels with excitation $\lambda =850\mathrm{nm}$ , $75\text{-}\mu \mathrm{m}$ $\left(512\mathrm{px}\right)$ image width, and $20\text{-}\mu \mathrm{m}$ distances between slices. We observed that too high laser intensities (20% of maximal power) led to plasma formation in the gel, while too low intensities $(<5\mathrm{\%})$ did not generate a reasonable signal-to-noise ratio in the backward detector. We thus operated the laser at 10% of maximal power. Image analysis was performed in ImageJ. Each optical section was first median filtered for noise reduction (radius 1). The threshold on each section was increased iteratively until $⩽5\mathrm{\%}$ of the pixels were left. This part of the macro was kindly provided by Jerome Mutterer, Strasbourg, France. For the pixels above threshold, the average intensity was calculated as a measure for SHG signal intensity. The average background signal was calculated from the $⩽5\mathrm{\%}$ of the pixels with the darkest gray values. Arrangement of all presented figures was performed in Adobe Photoshop CS2.

2.5.

Mouse Cremaster Preparation

Male C57BL/6 mice (age $10\text{to}12\text{weeks}$ ) were purchased from Charles River (Sulzfeld, Germany). Animals were housed under conventional conditions with free access to food and water. Tissue extraction was performed according to German legislation for the protection of animals. The animals were euthanized by an intraperitoneal pentobarbital overdose (Narcoren, Merial, Germany). Subsequently, the right cremaster muscle was exposed through a ventral incision of the scrotum. The muscle was opened ventrally, and epididymis and testicle were detached. The cremaster was explanted, placed on a standard microscopic glass slide, with or without mirror underneath, and covered with 0.9% NaCl. On two sides, the edges of the muscle were weighed down with coverslips to avoid floating of the tissue. These muscle preparations have a thickness of about $200\mu \mathrm{m}$ . 0.9% NaCl was used for immersion. Observation was performed in the central part, without coverslips between muscle and objective. Imaging was either with $1270\mathrm{nm}$ [at 80% total OPO power (i.e., about $200\mathrm{mW}$ behind the objective) with squared images, $1600\mathrm{px}$ , $400\mu \mathrm{m}$ ] or with $850\mathrm{nm}$ (2% TiSa Power, squared images, $512\mathrm{px}$ , $400\mu \mathrm{m}$ ).

3.1.

Fluorescence

To investigate whether two-photon-induced fluorescence signal intensity can be improved by an underlying mirror, we used two fluorescent test slides with different spectral properties. Z-scans were performed from top to bottom of the slides, with an uncoated control coverslip or a mirror (coverslip coated with aluminum) underneath. Intensity distributions showed the highest fluorescence near the surface of the slide (Fig. 1 ). Intensity dropped to less than half in the first $0.5\mathrm{mm}$ . Comparison with fluorescence signal intensity detected in the forward direction (data not shown) showed that this was due to absorption and/or scattering of excitation light and thus due to reduced occurrence of the two-photon effect.

Fig. 1

Fluorescence intensity (left $y$ axis) without (red circles) and with a mirror attached underneath a test slide (blue triangles). The ratio is shown as gray squares (right $y$ axis). (a) Blue test slide, average from five $z$ -stacks from different areas. (b) Red test slide, average from six $z$ -stacks from different areas. Standard deviation is indicated with error bars. However, variation was so small that error bars are mostly invisible. The nominal distances from the top of the 3-D stack are given ( $x$ axis). The first measured point $\left(0\mu \mathrm{m}\right)$ is above the slide. (Color online only.)

Comparison of fluorescence intensities obtained with and without mirrors revealed a strong effect close to the mirror, with signal intensities up to twice as high when the mirror was used (Fig. 1). The effect was gradually lost with increased distance from the mirror. At $400\text{-}\mu \mathrm{m}$ nominal distance away from the mirror, and thus an actual distance of $500\mu \mathrm{m}$ (see Sec. 2.3), the signal increase was still about 20%. While in the upper half of the slide, signal intensity decreased with increasing distance from the top surface, in the lower half, signal intensity first leveled out and then increased when approaching the mirror. This effect was more pronounced in the blue test slide [Fig. 1] than in the red test slide [Fig. 1], probably due to different absorption or scattering behavior of the slides at the respective wavelengths.

3.2.

Collagen I Matrix as an SHG-Generating Sample

To test the effect of an underlying mirror on SHG signal intensity, we selected a specimen with a regular three-dimensional distribution of harmonophores throughout a volume, a collagen I gel that was cast over a mirror and a control coverslip. Collagen is the most widely studied biological inductor of SHG signals (e.g., Refs. 6, 9, 20, 21). Visual inspection of SHG images from several positions of the gel confirmed even collagen fiber distribution. Illumination was with $850\mathrm{nm}$ , inducing SHG at $425\mathrm{nm}$ . 3-D stacks with an optical thickness of over a millimeter were recorded.

Imaging paths of forward and backward signals differed in important aspects (collection through objective versus condenser, shorter path for forward detection), thus a comparison of intensities does not necessarily reflect the amount of signal generated in the sample. With this limitation in mind, we found that the forward SHG signal [Fig. 2 ] had a higher intensity and a better signal-to-noise ratio than the conventional backward signal [Fig. 2], as previously described (see Sec. 1). Visual inspection showed that signals collected with the mirror [Fig. 2] did not reach the signal-to-noise ratio nor the intensity of forward signals. However, mirror-augmented signals had a better signal-to-noise ratio and were brighter than conventional backward signals that were recorded with the same detector and light path and are thus directly comparable.

Fig. 2

SHG signals in a collagen gel. (a) Parts of optical sections from a control stack at a depth of $60\mu \mathrm{m}$ (top) and $1200\mu \mathrm{m}$ (bottom) recorded with the forward detector. (b) Identical sections recorded with the backward detector. (c) Sections at the same depths from a stack recorded with underlying mirror. (d) Images as in (c), with thresholding of the brightest 5% pixels. Images in (b) and (c) were recorded with the same detector and processed identically. A different brightness adjustment had to be applied to (a) to avoid digital overexposure. Scale bar: $10\mu \mathrm{m}$ . (e) and (f) SHG signal intensity throughout the depth of a collagen gel. The average intensity of the brightest 5% pixels [see (d)] was measured. The average intensity of the darkest 5% (background) is given to allow an estimation of the detector baseline (dark curves). (e) Forward SHG (green); (f) backward SHG without mirror (red) and mirror-amplified SHG (blue) (Color online only.).

To confidently exclude background pixels from quantitative intensity measurements of SHG signals, we determined the brightest 5% of pixels in each section of the 3-D stack [Fig. 2] and calculated their average intensity. While this procedure will not include the complete SHG signals, it allows a reproducible intensity comparison throughout image stacks. Since the fraction of the incoming light that is scattered in the sample increases with depth, a decrease of two-photon-induced events and thus signal intensity can be expected in a regular sample. Accordingly, we observed a steady decrease of signal intensity from the top to the bottom of 3-D stacks when recorded backward without mirror as well as with the forward detector [Figs. 2 and 2]. However, the mirror-augmented SHG signal reached its maximal intensity close to the mirror, $1.2\mathrm{mm}$ away from the upper gel surface [Fig. 2]. The benefit of additional light collected with the mirror thus overcompensated the decrease in signal generation caused by the reduced occurrence of the two-photon effect deep in the gel. Compared to control stacks, increased signal intensity was detectable already $1\mathrm{mm}$ above the mirror.

3.3.

SHG and THG in the Mouse Cremaster Muscle

To explore whether benefits of mirror-augmented HHG signals would occur in an animal tissue, we analyzed explanted mouse cremaster muscles. This tissue contains collagen fibers and myosin. SHG by myosin^{21, 22, 23, 24} and THG by striated muscle tissue,²⁴ have been described. Since the molecular structure of striated muscle is very regular, we considered muscle fibers suitable to compare signal intensities under various conditions. When spread out, explanted muscles were up to $200\mu \mathrm{m}$ thick. To allow simultaneous recording of SHG and THG signals, we illuminated with $1270\mathrm{nm}$ to produce THG at $423\mathrm{nm}$ and SHG at $635\mathrm{nm}$ [Figs. 3, 3, 3, 3 ]. Since SHG and THG signal intensities are dependent on the direction of polarization of the incoming laser beam,²⁴ only muscle fibers that were parallel to the $x$ axis were evaluated.

Fig. 3

Higher harmonic imaging in the mouse cremaster muscle. (a) to (f) Illumination with $1270\mathrm{nm}$ . THG (a,c) and corresponding SHG (b,d) images recorded with the backward detector. At identical recording and image processing parameters, intensity was very low without mirror (a,b) while images collected with the help of the mirror (c,d) are already partially saturated. Note vertical shadows due to structures in other focal planes. (e) and (f) Quantitative analysis of signal intensity in muscle striations in THG (e) and SHG (f) image stacks. Individual intensity measurements in striated muscle are indicated by blue circles (with mirror) or red diamonds (control, no mirror). Averages are drawn as continuous lines. To allow the same scaling for both diagrams, two values in (f) had to be off chart; they are indicated numerically. (g) to (i) SHG with $860\text{-}\mathrm{nm}$ illumination detected in the forward direction (g); the same optical section detected with the backward detector (h) and an area from the same muscle with mirror-augmented signal (i). (h) and (i) were processed identically. Note the absence of SHG signal from myosin in the backward-detected image at the given settings. The mirror-augmented image clearly shows muscle striation. (Color online only.)

Rectangular regions of interest (ROIs) were set in the brightest striated muscle fiber areas of each optical section of SHG and THG image stacks, carefully avoiding other signals such as collagen fibers. Up to four ROIs were placed in each image frame, and their mean intensity was measured.

For THG, we found that all ROIs recorded with the backward detector and without a mirror in the light path $(n=141)$ had mean intensities below 46 (average 24, standard deviation 8.5), while all ROIs recorded with the mirror present $(n=249)$ had mean intensities above 56 (average 187, s.d. 66); the groups were thus nonoverlapping [Fig. 3]. On average, mirror-augmented THG signals were 7.6 times brighter than those from control images. For SHG, all control ROIs $(n=150)$ had mean intensities below 32 (average 15, s.d. 5), while all mirrored ROIs $(n=212)$ had intensities above 44 [average 147, s.d. 77; Fig. 3]. The intensity ratio between these two nonoverlapping groups was 9.6. Seven image stacks were evaluated for mirror-augmented images, and 10 for control images. Despite this, for both SHG and THG images, fewer ROIs could be defined in control images, since suitable sites with recognizable striation were often limited. We conclude that sites with low signal intensities in nonmirror control images dropped out of the evaluation and that the evaluation may thus be biased to underestimate the true intensity ratio between mirror-augmented and control image stacks. While in collagen gels, the signal intensity increased at a distance of less than $200\mu \mathrm{m}$ toward the mirror, in the cremaster mirror-augmented signal, intensities decreased slightly [Figs. 3 and 3], a likely consequence of stronger scattering in muscle tissue.

To compare “with mirror” and “no mirror” backward signals against forward signal, we induced SHG with $860\mathrm{nm}$ [Figs. 3, 3, 3]. Due to the setup of our microscope system, this comparison was not possible with $1270\text{-}\mathrm{nm}$ illumination. Signals from collagen fibers and myosin were brighter when recorded with the forward detector [Fig. 3] relative to control images collected with the backward detector without mirror [Fig. 3], as previously published (see Sec. 4). As for the collagen gel, “with mirror” images [Fig. 3] revealed brighter intensities and more structural detail than control backward signals.